Methods and compositions for inhibiting polyomavirus-associated pathology

ABSTRACT

Disclosed herein are methods of eliciting an immune response against a polyomavirus (for example, BKV serotype I (BKV-I), BKV serotype II (BKV-II), BKV serotype III (BKV-III) and/or BKV serotype IV (BKV-IV)) and methods of treating or inhibiting polyomavirus-associated pathology (such as polyomavirus-associated nephropathy, BKV-associated hemorrhagic cystitis, or JC virus-associated progressive multifocal leukoencephalopathy; PML). Further disclosed are immunogenic compositions of use in the disclosed methods. Also disclosed are methods of selecting an organ transplant donor and/or recipient including detecting whether the prospective donor and/or recipient has BKV serotype-specific (such as BKV serotype IV-specific) neutralizing antibodies.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a divisional of U.S. patent application Ser. No. 15/694,567,filed Sep. 1, 2017, which is a divisional of U.S. patent applicationSer. No. 14/233,582, filed Jan. 17, 2014, issued as U.S. Pat. No.9,764,022, on Sep. 19, 2017, which is the U.S. National Stage ofInternational Application No. PCT/US2012/047069, filed Jul. 17, 2012,which was published in English under PCT Article 21(2), which in turnclaims the benefit of U.S. Provisional Application No. 61/508,897, filedJul. 18, 2011, all of which are incorporated by reference herein intheir entirety.

FIELD

This disclosure relates to the field of immunology, more specifically tomethods and compositions for producing an immune response topolyomavirus, particularly BK polyomavirus.

BACKGROUND

The process of organ transplantation has been revolutionized since thefirst successful kidney transplant in identical twins more than fivedecades ago (Harrison et al., Surg. Forum 6:432-436, 1956; Merrill etal., J. Am Med. Assoc. 160:277-282, 1956). Since then, the use ofimmunosuppressants, such as cyclosporine, have improved the outcome oftransplants (Caine, Mt. Sinai J. Med. 54:465-466, 1987), but the processis still fraught with many challenges, such as the management of chronicand acute rejection, nephrotoxicity from immunosuppressant and antiviraldrugs, and avoiding reactivated (or novel) infectious agents that couldthreaten the graft. To balance these needs, clinical guidelines on themanagement of kidney transplant (Kasiske et al., Am. J. Transpl.9:S1-S155, 2009) generally suggest the use of an immunosuppressant andan anti-proliferative agent in the initial stages of the process,followed by a lowering of dose of immunosuppressants if there is noacute rejection. However, careful monitoring of allograft function iscrucial; and tests to detect increase in proteinuria, elevated serumcreatinine levels, and detection of viral nucleic acids in plasma arealso recommended.

One of the problems that threatens kidney allograft survival is thedevelopment of polyomavirus-associated nephropathy (PVAN) (Purighalla etal., Am. J. Kidney Dis. 26:671-673, 1995; also known as BKV associatednephropathy (BKVN)). Left untreated, PVAN can lead to a loss of theallograft, but early diagnosis, monitoring and intervention can preventit. In kidney transplant recipients, current estimates of PVAN are about1-10% (Ramos et al., Clin. Transpl. 2002:143-153; Hirsch et al.,Transplantation 79:1277-1286, 2005), and graft losses range from 10-100%(Hirsch and Steiger, Lancet Inf. Dis. 3:611-623, 2003) depending on thedrug regimen, monitoring, and interventions performed.Polyomavirus-associated pathologies such as PVAN or progressivemultifocal leukoencephalopathy (PML) also cause significant morbidity oreven mortality in other patients receiving immunosuppressive therapy(for example, for auto-immune disorders).

SUMMARY

Disclosed herein are methods of eliciting an immune response against apolyomavirus (for example, BKV serotype I (BKV-I), BKV serotype II(BKV-II), BKV serotype III (BKV-III), and/or BKV serotype IV (BKV-IV))and methods of treating or inhibiting polyomavirus-associated pathology(such as PVAN, BKV-associated hemorrhagic cystitis, or JCvirus-associated PML). Further disclosed are immunogenic compositions ofuse in the disclosed methods. In some embodiments, the immunogeniccomposition includes at least one capsid polypeptide (or a nucleic acidencoding such polypeptides) from two or more BKV serotypes (e.g., amultivalent immunogenic composition).

Also disclosed are methods of selecting a transplant donor and/ortransplant recipient (for example a renal transplant donor or recipient)including detecting whether the prospective donor and/or recipient hasBKV serotype-specific (such as BKV-IV-specific) neutralizing antibodies.

The foregoing and other features of the disclosure will become moreapparent from the following detailed description, which proceeds withreference to the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a series of graphs showing ELISA and neutralizing antibodytiters in mice immunized with virus-like particles (VLPs) formed byexpression of recombinant VP1 capsid proteins derived from BKV-I isolateKOM-5 (subtype BKV-Ib1) or BKV-IV isolate A-66H (subtype BKV-IVc2). Sixmice were immunized with BKV-I (circles) or BKV-IV (squares) VLPs. Inthe top panel, sera were titered using separate BKV-I (x axis) or BKV-IV(y axis) VLP ELISAs. A data point from one relatively non-responsiveanimal is shown as an open circle. The middle panel depicts BKVgenotype-specific neutralizing titers for the same set of mice. Thebottom panel shows the ratio of the neutralizing titer for the BKVgenotype administered as a vaccine versus the neutralizing titer for theheterologous BKV genotype for individual animals. The non-responsiveanimal was excluded from the analysis in the bottom panel. In the topand middle panels, the diagonal line shows a theoretical 1:1 correlationbetween BKV-I and BKV-IV titers.

FIG. 2 is a series of graphs showing BKV-I and BKV-IV ELISA versusneutralizing titers in VLP immunized mice. ELISA (x axis) orneutralizing titers (y axis) for six BKV-I (circles) or BKV-IV (squares)VLP immunized mice are shown. Neutralizing titers against the BKV-Ipseudovirus are shown in the left panel and anti-BKV-IV titers are shownin the right panel. A data point from the relatively non-responsiveanimal (FIG. 1 ) is shown as an open circle.

FIG. 3 is a pair of graphs showing BKV-I and BKV-IV serological titersin healthy adults. Sera from 48 healthy adults were evaluated for BKVtype-specific serological titers. The upper panel shows BKV-I and BKV-IVtiters evaluated by ELISA. The lower panel shows neutralizing titers.The diagonal line shows a theoretical 1:1 correlation between BKV-I andBKV-IV titers.

FIG. 4 is a series of graphs showing BKV-I and BKV-IV ELISA versusneutralizing titers in healthy adults. ELISA (x axis) or neutralizingtiters (y axis) for BKV-I (circles) or BKV-IV (squares) are shown.Neutralizing titers against the BKV-I pseudovirus are shown in the leftpanel and anti-BKV-IV titers are shown in the right panel.

FIG. 5 is a series of graphs showing BKV-I and BKV-IV serologicalpatterns in sera from individual kidney transplant recipients. Sera fromkidney transplant recipients were titered for the presence of BKV-I(circles) or BKV-IV (squares) type-specific neutralizing titers (yaxis). The neutralizing titer categories shown on they axis are definedas: 1)<95% neutralization at a serum dilution of 1:100; 2)>95%neutralization at 1:100; 3)>95% neutralization at 1:500; 4)>95%neutralization at 1:5000; and 5)>95% neutralization at 1:50,000. Serawere collected at five different time points (x axis) spanning roughly1, 4, 12, 26, and 52 weeks post-transplantation, designated A-E. In eachpanel, the notations in the bottom right corner represent the BKVgenotype (I or IV) observed in the patient's urine (superscript u) orblood (superscript b). The subject denoted I/IV^(u) showed urinaryshedding of BKV-I at week 5 and urinary shedding of BKV-IV at week 16.The patterns of 12 representative patients are shown.

FIG. 6 is a series of graphs showing BKV-I and BKV-IV serologicalprofiles in all 108 kidney transplant patients analyzed. Neutralizingtiter categories (y-axis) and time points (x-axis) are as described forFIG. 5 . The numbers at the top of each graph denote quantitation of BKVviruria (log 10 BKV DNA copies per ml) at each time point. Dashesindicate that BKV DNA was not detected in the urine. The symbol “nr”indicates no results for the time point. The symbol “utq” indicates thatthe BKV viruria signal was too low for accurate quantitation. Asterisksmark time points at which BKV viremia was quantitated. The symbol JC+indicates that JC virus DNA was detected.

FIG. 7 is a pair of graphs showing BKV-I and BKV-IV neutralizing titersin kidney transplant recipients at study entry and exit. Sera from 108kidney transplant recipients were titered for the presence of BKV-I (toppanel) or BKV-IV (bottom panel) type-specific neutralizing antibodies.The percentages of patients at a particular titer cut-off at study entry(one week after transplantation) are depicted as open bars, while thepercentages of patients at a particular titer cut-off at study exit (oneyear post-transplantation) are depicted as filled bars.

FIG. 8 is a sequence alignment of BKV VP1 polypeptides. Because it isnot possible to distinguish between Ia and Ib1 subtypes based on VP1amino acid sequences, BKV-Ia indicates genotypes Ia/Ib1 and BKV-Ibindicates genotype Ib2. It is also not possible to distinguish betweenBKV-IV subtypes based on VP1 amino acid sequences. Therefore, BKV-IVindicates genotypes IV-b1/IV-c2. Amino acids that are completelyconserved among all BKV types are shaded. Sequence identifiers for theamino acid sequences are provided in Table 5 (below).

FIG. 9 is a phylogenetic tree showing the relationship of additionalpartial BKV VP1 sequences with selected BKV-I to BKV-IV VP1 sequences

FIG. 10 is a sequence alignment of BKV partial VP1 polypeptides (SEQ IDNOs: 126-158) with amino acids 31-174 of BKV-Ia (SEQ ID NO; 52), BKV-Ia(SEQ ID NO: 75), BKV-Ib2 (SEQ ID NO: 75), BKV-Ic (SEQ ID NO: 103),BKV-II (SEQ ID NO: 105), BKV-III (SEQ ID NO: 107), BKV-IVb1 (SEQ ID NO:125), and BKV-IVc2 (SEQ ID NO: 124) polypeptides.

SEQUENCE LISTING

Any nucleic acid and amino acid sequences listed herein or in theaccompanying sequence listing are shown using standard letterabbreviations for nucleotide bases and amino acids, as defined in 37C.F.R. 1.822. In at least some cases, only one strand of each nucleicacid sequence is shown, but the complementary strand is understood asincluded by any reference to the displayed strand.

The Sequence Listing is submitted as an XML file in the form of the filenamed 4239-87376-47_Sequence_Listing.xml, which was created on Aug. 25,2022, and is 207,933 bytes, which is incorporated by reference herein.

SEQ ID NOs: 1-3 are amino acid sequences of exemplary BKV-Ib1 VP1, VP2,and VP3 proteins, respectively.

SEQ ID NOs: 4-6 are amino acid sequences of exemplary BKV-IVc2 VP1, VP2,and VP3 proteins, respectively.

SEQ ID NOs: 7-9 are amino acid sequences of exemplary BKV-II VP1, VP2,and VP3 proteins, respectively.

SEQ ID NOs: 10-12 are amino acid sequences of exemplary BKV-III VP1,VP2, and VP3 proteins, respectively.

SEQ ID NO: 13 is an exemplary BKV-Ia VP1 amino acid sequence.

SEQ ID NO: 14 is an exemplary BKV-Ib2 VP1 amino acid sequence.

SEQ ID NO: 15 is an exemplary BKV-Ic VP1 amino acid sequence.

SEQ ID NO: 16 is an exemplary BKV-IV-b1 VP1 amino acid sequence.

SEQ ID NOs: 17-19 are amino acid sequences of exemplary JCV-1A VP1, VP2,and VP3 proteins, respectively.

SEQ ID NO: 20 is an exemplary JCV-2A VP1 amino acid sequence.

SEQ ID NO: 21 is an exemplary JCV-3B VP1 amino acid sequence.

SEQ ID NOs: 22-23 are exemplary JCV consensus VP2 and VP3 amino acidsequences, respectively.

SEQ ID NOs: 24-26 are exemplary BKV-Ib1 VP1, VP2, and VP3 encodingnucleic acid sequences, respectively.

SEQ ID NOs: 27-29 are exemplary BKV-IVc2 VP1, VP2, and VP3 encodingnucleic acid sequences, respectively.

SEQ ID NOs: 30-32 are exemplary BKV-II VP1, VP2, and VP3 encodingnucleic acid sequences, respectively.

SEQ ID NOs: 33-35 are exemplary BKV-III VP1, VP2, and VP3 encodingnucleic acid sequences, respectively.

SEQ ID NOs: 36-38 are exemplary JCV-1A VP1, VP2, and VP3 encodingnucleic acid sequences, respectively.

SEQ ID NOs: 39-41 are exemplary codon-optimized BKV-IVc2 VP1, VP2, andVP3 polypeptide encoding nucleic acid sequences, respectively.

SEQ ID NO: 42 is an exemplary codon-optimized BKV-Ia VP1 polypeptideencoding nucleic acid sequence.

SEQ ID NO: 43 is an exemplary codon-optimized BKV-Ib2 VP1 polypeptideencoding nucleic acid sequence.

SEQ ID NO: 44 is an exemplary codon-optimized BKV-Ic VP1 polypeptideencoding nucleic acid sequence.

SEQ ID NOs: 45 and 46 are exemplary codon-optimized BKV-II and BKV-IIIVP1 polypeptide encoding nucleic acid sequences, respectively.

SEQ ID NO: 47 is an exemplary codon-optimized BKV-IVb1 VP1 polypeptideencoding nucleic acid sequence.

SEQ ID NO: 48 is an exemplary codon-optimized JCV-2A VP1 polypeptideencoding nucleic acid sequence.

SEQ ID NO: 49 is an exemplary codon-optimized JCV-3B VP1 polypeptideencoding nucleic acid sequence.

SEQ ID NOs: 50 and 51 are exemplary codon-optimized JCV consensus VP2and VP3 polypeptide encoding nucleic acid sequences, respectively.

SEQ ID NOs: 52-125 are exemplary BKV VP1 polypeptide amino acidsequences.

SEQ ID NOs: 126-160 are exemplary partial BKV VP1 polypeptide amino acidsequences.

DETAILED DESCRIPTION

BKV is a ubiquitous DNA virus and up to 90% of healthy individuals areseropositive. This virus is believed to initiate infection in theurinary tract and then remain latent without disturbing its host, withoccasional reactivation in the form of low-level shedding of virions inthe urine (viruria). However, in immunocompromised individuals BKV (andthe related JC polyomavirus) can cause significant morbidity or evenmortality.

In pediatric kidney transplants, being seronegative for BKV by aserological test before the procedure has been associated withdeveloping PVAN. In adult kidney transplant recipients, it has beensuggested that the role of donor BKV status plays a role in developingPVAN. However, pre-transplant BKV serology is not usually monitored;both because nearly all adults are believed to be seropositive for BKV,and because it has been generally believed that seropositivity for BKVis associated with protection against development of PVAN. Furthermore,the role of intravenous immunoglobulin (IVIG) in treating PVAN has beenunclear.

BKV consists of four subgroups (or types; BKV-I, BKV-II, BKV-III, andBKV-IV) that have been equated with separate serotypes and have limitedcross-reactivity as measured by hemagglutination inhibition (HI) andneutralization assays. However, the high BKV prevalence rates in humans,as measured by polymerase chain reaction or BKV seropositivity generallyrefer to serotype I only. The incidence of the other BKV types inpatients prior to kidney or bone marrow transplant in Caucasians hasbeen estimated to be about 3% for BKV-II, 6-7% for BKV-IV andundetectable for BKV-III. However, the present inventors have found apanel of sera from 48 healthy adults to be substantially higher thanexpected for genotypes II, III, and IV. In a few cases, the inventorshave demonstrated that kidney transplant patients who initially had onlyBKV-I viruria later developed viruria and viremia (presence of virus inthe bloodstream) with BKV-IV.

The cross-reactivity of anti-BKV antibodies between types has not beenrevisited since 1989; and it may have important implications in thedevelopment of PVAN in transplant patients, especially if the organdonor is positive for a less common BKV type. The present inventors havedemonstrated that 23-43% of renal transplant patients with undetectablelevels of BKV-IV neutralizing antibodies at the time of transplantseroconverted (changed from a negative result to a positive result in aserologic test) within one year, irrespective of their BKV-I serostatus.The small number of initially BKV-I seronegative renal transplantrecipients all seroconverted for BKV-I, irrespective of their BKV-IVserostatus.

Thus, the inventors have demonstrated that presence of neutralizingantibodies against one BKV serotype does not protect from infection withother BKV serotypes, as was previously believed. Furthermore, thepresence of neutralizing antibodies to BKV-Ib2 does not provideneutralization of BKV-Ia, demonstrating that not all BKV-I neutralizingantibodies can provide protection from all BKV-I subtypes. Thisindicates that vaccination of individuals (such as organ transplantrecipients or other immunocompromised individuals) with a vaccine to asingle BKV serotype or subtype may not effectively elicit an immuneresponse to all serotypes or subtypes and may not provide adequateprotection from polyomavirus-associated pathologies such as PVAN or PML.Furthermore, it indicates that the prevalence of BKV-I in the generalpopulation does not protect at risk individuals from infection withother BKV serotypes (such as BKV-IV).

I. Abbreviations

BKV BK polyomavirus

BKV-I BKV serotype I

BKV-II BKV serotype II

BKV-III BKV serotype III

BKV-IV BKV serotype IV

ELISA enzyme-linked immunosorbant assay

IVIG intravenous immunoglobulin

JCV JC polyomavirus

PML progressive multifocal leukoencephalopathy

PVAN polyomavirus-associated nephropathy

SV40 simian virus 40

VLP virus-like particle

II. Terms

Unless otherwise noted, technical terms are used according toconventional usage. Definitions of common terms in molecular biology maybe found in Benjamin Lewin, Genes VII, published by Oxford UniversityPress, 2000 (ISBN 019879276X); Kendrew et al. (eds.), The Encyclopediaof Molecular Biology, published by Blackwell Publishers, 1994 (ISBN0632021829); Robert A. Meyers (ed.), Molecular Biology andBiotechnology: a Comprehensive Desk Reference, published by Wiley, John& Sons, Inc., 1995 (ISBN 0471186341); and George P. Rédei, EncyclopedicDictionary of Genetics, Genomics, and Proteomics, 2nd Edition, 2003(ISBN: 0-471-26821-6).

The following explanations of terms and methods are provided to betterdescribe the present disclosure and to guide those of ordinary skill inthe art to practice the present disclosure. The singular forms “a,”“an,” and “the” refer to one or more than one, unless the contextclearly dictates otherwise. For example, the term “comprising apolypeptide” includes single or plural polypeptides and is consideredequivalent to the phrase “comprising at least one polypeptide.” As usedherein, “comprises” means “includes.” Thus, “comprising A or B,” means“including A, B, or A and B,” without excluding additional elements.

All publications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety for allpurposes. In case of conflict, the present specification, includingexplanations of terms, will control.

Although methods and materials similar or equivalent to those describedherein can be used to practice or test the disclosed technology,suitable methods and materials are described below. The materials,methods, and examples are illustrative only and not intended to belimiting.

To facilitate review of the various embodiments of this disclosure, thefollowing explanations of specific terms are provided:

Antibody: A protein (or protein complex) that includes one or morepolypeptides substantially encoded by immunoglobulin genes or fragmentsof immunoglobulin genes. The recognized immunoglobulin genes include thekappa, lambda, alpha, gamma, delta, epsilon, and mu constant regiongenes, as well as the myriad of immunoglobulin variable region genes.Light chains are classified as either kappa or lambda. Heavy chains areclassified as gamma, mu, alpha, delta, or epsilon, which in turn definethe immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively. Aneutralizing antibody is an antibody which, on mixture with thehomologous infectious agent (such as a polyomavirus), reduces theinfectious titer. In some examples, a neutralizing antibody is anantibody that blocks the ability of its antigen to perform aphysiological function.

The basic immunoglobulin (antibody) structural unit is generally atetramer. Each tetramer is composed of two identical pairs ofpolypeptide chains, each pair having one “light” (about 25 kDa) and one“heavy” (about 50-70 kDa) chain. The N-terminus of each chain defines avariable region of about 100 to 110 or more amino acids primarilyresponsible for antigen recognition. The terms “variable light chain”(V_(L)) and “variable heavy chain” (V_(H)) refer, respectively, to theselight and heavy chains.

As used herein, the term “antibodies” includes intact immunoglobulins aswell as a number of well-characterized fragments. For instance, Fabs,Fvs, and single-chain Fvs (SCFvs) that bind to target protein (orepitope within a protein or fusion protein) would also be specificbinding agents for that protein (or epitope). These antibody fragmentsare defined as follows: (1) Fab, the fragment which contains amonovalent antigen-binding fragment of an antibody molecule produced bydigestion of whole antibody with the enzyme papain to yield an intactlight chain and a portion of one heavy chain; (2) Fab′, the fragment ofan antibody molecule obtained by treating whole antibody with pepsin,followed by reduction, to yield an intact light chain and a portion ofthe heavy chain; two Fab′ fragments are obtained per antibody molecule;(3) (Fab′)₂, the fragment of the antibody obtained by treating wholeantibody with the enzyme pepsin without subsequent reduction; (4)F(ab′)₂, a dimer of two Fab′ fragments held together by two disulfidebonds; (5) Fv, a genetically engineered fragment containing the variableregion of the light chain and the variable region of the heavy chainexpressed as two chains; and (6) single chain antibody, a geneticallyengineered molecule containing the variable region of the light chain,the variable region of the heavy chain, linked by a suitable polypeptidelinker as a genetically fused single chain molecule. Methods of makingthese fragments are routine (see, for example, Harlow and Lane, UsingAntibodies: A Laboratory Manual, CSHL, New York, 1999).

BK polyomavirus (BKV): A polyomavirus originally isolated from patientB.K. after renal transplantation (Gardner et al., Lancet 1:1253-1257,1971). Four BKV serotypes are known (serotypes I-IV; e.g., Knowles etal., J. Med. Virol. 28:118-123, 1989). BKV is nearly ubiquitous, and upto 90% of healthy individuals are seropositive for BKV. Acute infectionis generally asymptomatic and proceeds to latent infection, primarily inthe urogenital tract. BKV can be reactivated in immunocompromisedindividuals, and can cause significant morbidity, particularly in renaltransplant patients.

BKV nucleic acid and amino acid sequences are publicly available. Forexample, GenBank Accession Nos. AB211374, AB263920, AB211386, andAB369093 disclose exemplary BKV-I, BKV-II, BKV-III, and BKV-IV nucleicacid sequences, respectively, all of which are incorporated by referenceas present in GenBank on Jul. 15, 2011.

Capsid polypeptide: One of three structural proteins that forms thepolyomavirus capsid. The polyomavirus capsid is formed from viralprotein 1 (VP1), viral protein 2 (VP2), and viral protein 3 (VP3).

Codon-optimized: A “codon-optimized” nucleic acid refers to a nucleicacid sequence that has been altered such that the codons are optimal forexpression in a particular system (such as a particular species or groupof species). For example, a nucleic acid sequence can be optimized forexpression in mammalian cells, bacteria or yeast. Codon optimizationdoes not alter the amino acid sequence of the encoded protein.

Conservative variants: A substitution of an amino acid residue foranother amino acid residue having similar biochemical properties.“Conservative” amino acid substitutions include those substitutions thatdo not substantially affect or decrease an activity or antigenicity of apolypeptide. A peptide can include one or more amino acid substitutions,for example 1-10 conservative substitutions, 2-5 conservativesubstitutions, 4-9 conservative substitutions, such as 1, 2, 5 or 10conservative substitutions. Specific, non-limiting examples of aconservative substitution include the following examples (Table 1).

TABLE 1 Exemplary conservative amino acid substitutions Original AminoAcid Conservative Substitutions Ala Ser Arg Lys Asn Gln, His Asp Glu CysSer Gln Asn Glu Asp His Asn; Gln Ile Leu, Val Leu Ile; Val Lys Arg; Gln;Glu Met Leu; Ile Phe Met; Leu; Tyr Ser Thr Thr Ser Trp Tyr Tyr Trp; PheVal Ile; Leu

The term conservative variation also includes the use of a substitutedamino acid in place of an unsubstituted parent amino acid, provided thatantibodies raised to the substituted polypeptide also immunoreact withthe unsubstituted polypeptide, or that an immune response can begenerated against the substituted polypeptide that is similar to theimmune response against the unsubstituted polypeptide. Thus, in oneembodiment, non-conservative substitutions are those that reduce anactivity or antigenicity.

Immune response: A response of a cell of the immune system, such as aB-cell, T-cell, macrophage or polymorphonucleocyte, to a stimulus suchas an antigen. An immune response can include any cell of the bodyinvolved in a host defense response, for example, an epithelial cellthat secretes an interferon or a cytokine. An immune response includes,but is not limited to, an innate immune response or inflammation.

Immunocompromised: An immunocompromised subject is a subject who isincapable of developing or unlikely to develop a robust immune response,usually as a result of disease, malnutrition, or immunosuppressivetherapy. An immunocompromised immune system is an immune system that isfunctioning below normal Immunocompromised subjects are more susceptibleto opportunistic infections, for example viral, fungal, protozoan, orbacterial infections, prion diseases, and certain neoplasms.

Those who can be considered to be immunocompromised include, but are notlimited to, subjects with AIDS (or HIV positive), subjects with severecombined immunodeficiency (SCID), diabetics, subjects who have hadtransplants and who are taking immunosuppressants, and those who arereceiving chemotherapy for cancer. Immunocompromised individuals alsoincludes subjects with most forms of cancer (other than skin cancer),sickle cell anemia, cystic fibrosis, those who do not have a spleen,subjects with end stage kidney disease (dialysis), and those who havebeen taking corticosteroids or other immune suppressing therapy on afrequent basis within the last year.

Immunosuppressant: Any compound that decreases the function or activityof one or more aspects of the immune system, such as a component of thehumoral or cellular immune system or the complement system.Immunosuppressants are also referred to as “immunosuppressive agents” or“immunosuppressive therapies.”

In some examples, an immunosuppressant includes, but is not limited to:(1) antimetabolites, such as purine synthesis inhibitors (such asinosine monophosphate dehydrogenase (IMPDH) inhibitors, e.g.,azathioprine, mycophenolate, and mycophenolate mofetil), pyrimidinesynthesis inhibitors (e.g., leflunomide and teriflunomide), andantifolates (e.g., methotrexate); (2) calcineurin inhibitors, such astacrolimus, cyclosporine A, pimecrolimus, and voclosporin; (3) TNF-αinhibitors, such as thalidomide and lenalidomide; (4) IL-1 receptorantagonists, such as anakinra; (5) mammalian target of rapamycin (mTOR)inhibitors, such as rapamycin (sirolimus), deforolimus, everolimus,temsirolimus, zotarolimus, and biolimus A9; (6) corticosteroids, such asprednisone; and (7) antibodies to any one of a number of cellular orserum targets (including anti-lymphocyte globulin and anti-thymocyteglobulin).

Exemplary cellular targets and their respective inhibitor compoundsinclude, but are not limited to complement component 5 (e.g.,eculizumab); tumor necrosis factors (TNFs) (e.g., infliximab,adalimumab, certolizumab pegol, afelimomab and golimumab); IL-5 (e.g.,mepolizumab); IgE (e.g., omalizumab); BAYX (e.g., nerelimomab);interferon (e.g., faralimomab); IL-6 (e.g., elsilimomab); IL-12 andIL-13 (e.g., lebrikizumab and ustekinumab); CD3 (e.g., muromonab-CD3,otelixizumab, teplizumab, visilizumab); CD4 (e.g., clenoliximab,keliximab and zanolimumab); CD11a (e.g., efalizumab); CD18 (e.g.,erlizumab); CD20 (e.g., rituximab, afutuzumab, ocrelizumab,pascolizumab); CD23 (e.g., lumiliximab); CD40 (e.g., teneliximab,toralizumab); CD52 (e.g., alemtuzumab); CD62L/L-selectin (e.g.,aselizumab); CD80 (e.g., galiximab); CD147/basigin (e.g., gavilimomab);CD154 (e.g., ruplizumab); BLyS (e.g., belimumab); CTLA-4 (e.g.,ipilimumab, tremelimumab); CAT (e.g., bertilimumab, lerdelimumab,metelimumab); integrin (e.g., natalizumab); IL-6 receptor (e.g.,tocilizumab); LFA-1 (e.g., odulimomab); and IL-2 receptor/CD25 (e.g.,basiliximab, daclizumab, inolimomab).

Inhibiting or treating a disease: “Inhibiting” a disease refers toinhibiting the full development of a disease, for example, PVAN, PML, orBKV-associated hemorrhagic cystitis. Inhibition of a disease can spanthe spectrum from partial inhibition to substantially completeinhibition (e.g., including, but not limited to prevention) of thedisease. In some examples, the term “inhibiting” refers to reducing ordelaying the onset or progression of a disease. A subject to beadministered with a therapeutically effective amount of the disclosedimmunogenic compositions can be identified by standard diagnosingtechniques for such a disorder, for example, basis of family history, orrisk factor to develop the disease or disorder. “Treatment” refers to atherapeutic intervention that ameliorates a sign or symptom of a diseaseor pathological condition after it has begun to develop.

Isolated: An “isolated” or “purified” biological component (such as anucleic acid, peptide, protein, protein complex, or virus-like particle)has been substantially separated, produced apart from, or purified awayfrom other biological components in the cell of the organism in whichthe component naturally occurs, that is, other chromosomal andextrachromosomal DNA and RNA, and proteins. Nucleic acids, peptides andproteins that have been “isolated” or “purified” thus include nucleicacids and proteins purified by standard purification methods. The termalso embraces nucleic acids, peptides and proteins prepared byrecombinant expression in a host cell, as well as chemically synthesizednucleic acids or proteins.

The term “isolated” or “purified” does not require absolute purity;rather, it is intended as a relative term. Thus, for example, anisolated biological component is one in which the biological componentis more enriched than the biological component is in its naturalenvironment within a cell, or other production vessel. Preferably, apreparation is purified such that the biological component represents atleast 50%, such as at least 70%, at least 90%, at least 95%, or greater,of the total biological component content of the preparation.

JC polyomavirus (JCV): A polyomavirus originally isolated from a patient(J.C.) with progressive multifocal leukoencephalopathy (Padgett et al.,Lancet 1:1257-1260, 1971). JCV is genetically similar to BKV and simianvirus 40 (SV40). JCV is very common in the general population, with70-90% of individuals seropositive for JCV. The initial site ofinfection may be the tonsils or gastrointestinal tract. The primarysites of JC infection are thought to be tubular epithelial cells in thekidney, the lining of the ureters and bladder, and oligodendrocytes andastrocytes in the central nervous system.

JCV can reactivate in immunocompromised individuals and can causeJCV-associated progressive multifocal leukoencephalopathy (PML), whichis usually fatal. PML occurs in about 10% of patients suffering fromHIV-induced AIDS and can also occur in other immunosuppressed patients,including but not limited to patients treated with rituximab,natalizumab, alemtuzumab, or efalizumab. JCV can also cause urinarytract pathology in some organ transplant recipients.

JCV nucleic acid and amino acid sequences are publicly available. Forexample, GenBank Accession Nos. NC_001699, AB038251, and AF281600disclose exemplary JCV nucleic acid sequences, all of which areincorporated by reference as present in GenBank on Jul. 15, 2011. JCVisolates have been classified into eight distinct genotypes, based inpart on the amino acid sequences of VP1 proteins of individual isolates(Cubitt et al., J. Neurovirol. 7:339-344, 2001).

Pharmaceutically acceptable carrier: The pharmaceutically acceptablecarriers (vehicles) useful in this disclosure are conventional.Remington: The Science and Practice of Pharmacy, The University of theSciences in Philadelphia, Editor, Lippincott, Williams, & Wilkins,Philadelphia, Pa., 21^(st) Edition (2005), describes compositions andformulations suitable for pharmaceutical delivery of one or moretherapeutic compositions, such as one or more polyomavirus capsidpolypeptides or fragments thereof, and additional pharmaceutical agents.

In general, the nature of the carrier will depend on the particular modeof administration being employed. For instance, parenteral formulationsusually comprise injectable fluids that include pharmaceutically andphysiologically acceptable fluids such as water, physiological saline,balanced salt solutions, aqueous dextrose, glycerol or the like as avehicle. For solid compositions (for example, powder, pill, tablet, orcapsule forms), conventional non-toxic solid carriers can include, forexample, pharmaceutical grades of mannitol, lactose, starch, ormagnesium stearate. In addition to biologically-neutral carriers,pharmaceutical compositions to be administered can contain minor amountsof non-toxic auxiliary substances, such as wetting or emulsifyingagents, preservatives, and pH buffering agents and the like, for examplesodium acetate or sorbitan monolaurate.

Polyomavirus: A genus of nonenveloped viruses having an icosahedralcapsid. The genome of polyomaviruses includes non-structural proteins(large T-antigen and small t-antigen), a non-coding region including anorigin of replication and promoters, and structural proteins (VP1, VP2,and VP3). Polyomaviruses include but are not limited to BK polyomavirus,JC polyomavirus, Merkel cell polyomavirus, and simian virus 40 (SV40).Related human polyomaviruses WU virus (Gaynor et al., PLoS Pathog.3:e64, 2007) and KI virus (Allander et al., J. Virol. 81:4130-4136,2007) have recently been reported in clinical samples

Polyomavirus infection is generally asymptomatic in healthy subjects.However, polyomavirus infection can occur or be reactivated inimmunocompromised individuals and can cause significant morbidity.

Polyomavirus-associated nephropathy (PVAN; also called BKpolyomavirus-associated nephropathy or BK virus nephritis) occurs in upto 10% of renal transplant recipients and is believed to be caused byBKV infection or reactivation of latent BKV infection. It causes kidneyallograft dysfunction and may lead to loss of the allograft.Polyomavirus-associated hemorrhagic cystitis is characterized byinflammation of the bladder leading to dysuria, hematuria, andhemorrhage. It can occur in bone marrow transplant recipients and otherindividuals who are receiving immunosuppressants or other therapieswhich decrease immune system function.

Sequence identity: The similarity between two nucleic acid sequences, ortwo amino acid sequences, is expressed in terms of the similaritybetween the sequences, otherwise referred to as sequence identity.Sequence identity is frequently measured in terms of percentage identity(or similarity or homology); the higher the percentage, the more similarthe two sequences are.

Methods of alignment of sequences for comparison are well known in theart. Various programs and alignment algorithms are described in: Smith &Waterman, Adv. Appl. Math. 2: 482, 1981; Needleman & Wunsch, J. Mol.Biol. 48: 443, 1970; Pearson & Lipman, Proc. Natl. Acad. Sci. USA 85:2444, 1988; Higgins & Sharp, Gene, 73: 237-244, 1988; Higgins & Sharp,Comput. Appl. Biosci. 5: 151-153, 1989; Corpet et al., Nucl. Acids Res.16, 10881-90, 1988; Huang et al., Comput. Appl. Biosci. 8, 155-65, 1992;and Pearson, Methods Mol. Biol. 24:307-331, 1994. Altschul et al. (J.Mol. Biol. 215:403-410, 1990) presents a detailed consideration ofsequence alignment methods and homology calculations.

The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J.Mol. Biol. 215:403-410, 1990) is available from several sources,including the National Center for Biotechnology Information (NCBI,Bethesda, Md.) and on the Internet, for use in connection with thesequence analysis programs blastp, blastn, blastx, tblastn and tblastx.By way of example, for comparisons of amino acid sequences of greaterthan about 30 amino acids, the Blast 2 sequences function is employedusing the default BLOSUM62 matrix set to default parameters (gapexistence cost of 11, and a per residue gap cost of 1). When aligningshort peptides (fewer than around 30 amino acids), the alignment isperformed using the Blast 2 sequences function, employing the PAM30matrix set to default parameters (open gap 9, extension gap 1penalties).

Nucleic acid sequences that do not show a high degree of sequenceidentity may nevertheless encode similar amino acid sequences, due tothe degeneracy of the genetic code. It is understood that changes innucleic acid sequence can be made using this degeneracy to producemultiple nucleic acid molecules that all encode substantially the sameprotein.

Subject: Living multi-cellular vertebrate organisms, a category thatincludes both human and non-human mammals (such as mice, rats, rabbits,sheep, horses, cows, and non-human primates).

Therapeutically effective amount: A quantity of a specified agentsufficient to achieve a desired effect in a subject being treated withthat agent. For example, this may be the amount of a polyomavirus capsidpolypeptide or nucleic acid (or fragment thereof) useful for elicitingan immune response in a subject and/or for inhibiting or preventinginfection or pathology by a polyomavirus (such as BKV or JCV). Ideally,in the context of the present disclosure, a therapeutically effectiveamount of a polyomavirus polypeptide or nucleic acid (or fragmentthereof) is an amount sufficient to increase resistance to, prevent,ameliorate, and/or treat infection caused by a polyomavirus in a subjectwithout causing a substantial cytotoxic effect in the subject. Theeffective amount of a polyomavirus polypeptide or nucleic acid (orfragment thereof) useful for increasing resistance to, preventing,ameliorating, and/or treating infection in a subject will be dependenton, for example, the subject being treated, the manner of administrationof the therapeutic composition, and other factors.

Virus-like particle (VLP): A non-replicating viral shell, derived fromany of several viruses. VLPs are generally composed of one or more viralproteins, such as, but not limited to, those proteins referred to ascapsid, coat, shell, surface and/or envelope proteins, orparticle-forming polypeptides derived from these proteins. VLPs can formspontaneously upon recombinant expression of the protein in anappropriate expression system. Methods for producing particular VLPs areknown in the art. The presence of VLPs following recombinant expressionof viral proteins can be detected using conventional techniques known inthe art, such as by electron microscopy, biophysical characterization,and the like. See, for example, Baker et al. (1991) Biophys. J.60:1445-1456; Hagensee et al. (1994) J. Virol. 68:4503-4505. Forexample, VLPs can be isolated by density gradient centrifugation and/oridentified by characteristic density banding. Alternatively,cryoelectron microscopy can be performed on vitrified aqueous samples ofthe VLP preparation in question, and images recorded under appropriateexposure conditions.

III. Immune Response to Polyomavirus

Disclosed herein are methods of eliciting an immune response against apolyomavirus (for example, BKV or JCV). The disclosed methods utilizeone or more capsid polypeptides (or a fragment thereof) of apolyomavirus to elicit an immune response in a subject. In someexamples, the methods are of use to treat, inhibit, or even preventinfection of a subject with a polyomavirus or to treat, inhibit, or evenprevent polyomavirus-associated disorders (for example, PVAN,BKV-associated hemorrhagic cystitis, and/or JCV-associated PML). In someembodiments, the methods include administering at least one capsidpolypeptide (or a fragment thereof) from each of two or more BKVserotypes (such as a multivalent BKV serotype immunogenic composition)to a subject. In some embodiments, the multivalent immunogeniccomposition includes at least one capsid polypeptide (or a fragmentthereof) from two or more BKV-I subtypes (such as BKV-Ia, BKV-Ib1,BKV-Ib2, and/or BKV-Ic subtypes). In additional embodiments, the methodsfurther include administering at least one JCV capsid polypeptide (or afragment thereof) to the subject. Also disclosed are methods ofidentifying a transplant donor and/or transplant recipient (such as arenal transplant donor or recipient) who does not have antibodies forone or more BKV serotypes (for example, BKV-IV or BKV-I), such as adonor and/or recipient who does not have detectable levels of antibodiescapable of neutralizing one or more BKV serotypes (for example, BKV-I orBKV-IV).

A. Methods of Eliciting an Immune Response to BKV

In some embodiments, the methods include eliciting an immune responseagainst a BKV in a subject (such as one or more BKV serotypes). Themethods include administering to a subject in need of enhanced immunityto BKV a therapeutically effective amount of at least one isolated BKV-Icapsid polypeptide (or a fragment thereof) or a nucleic acid encodingthe at least one BKV-I capsid polypeptide (such as at least one BKV-IaVP1 polypeptide, BKV-Ib1 VP1 polypeptide, BKV-Ib2 VP1 polypeptide,and/or BKV-Ic VP1 polypeptide) and a therapeutically effective amount ofat least one isolated BKV-IV capsid polypeptide (or a fragment thereof)or a nucleic acid encoding the at least one BKV-IV capsid polypeptide(such as at least one BKV-IVb1 VP1 polypeptide and/or BKV-IVc2 VP1polypeptide). In some examples, the at least one BKV-I capsidpolypeptide (or fragment thereof) and the at least one BKV-IV capsidpolypeptide (or fragment thereof) are different from one another. TheBKV-I and BKV-IV capsid polypeptides include one or more of VP1, VP2,and VP3 (such as SEQ ID NOs: 1-6 and 13-16), and are discussed in detailin Section IV, below. In particular examples, administering the at leastone isolated BKV-I capsid polypeptide includes administering a VLPincluding the BKV-I capsid polypeptide(s) and/or administering the atleast one BKV-IV capsid polypeptide includes administering a VLPincluding the BKV-IV capsid polypeptide(s). In particular examples, thesubject does not have BKV-IV neutralizing antibodies. In other examples,the subject does not have BKV-I neutralizing antibodies.

In one non-limiting example, the methods include administering to asubject in need of enhanced immunity to BKV a therapeutically effectiveamount of at least one BKV-Ia VP1 polypeptide, at least one BKV-Ib2 VP1polypeptide, and at least one BKV-IV VP1 polypeptide (such as at leastone BKV-IVb1 VP1 polypeptide and/or BKV-IVc2 VP1 polypeptide). In otherexamples, the methods may also include administering to a subject inneed of enhanced immunity to BKV a therapeutically effective amount ofat least one BKV-Ic VP1 polypeptide. In some examples, the BKV-Ia/Ib1VP1 polypeptide includes a glutamic acid at amino acid position 73and/or a glutamic acid at amino acid position 82 (such as SEQ ID NO: 1or SEQ ID NO: 13). In additional examples, the BKV-Ib2 VP1 polypeptideincludes a lysine residue at amino acid position 73 and/or an asparticacid at amino acid position 82 (such as SEQ ID NO: 14).

In some examples, the methods further include administering to thesubject a therapeutically effective amount of at least one isolatedBKV-II capsid polypeptide (or a fragment thereof) or a nucleic acidencoding the at least one BKV-II capsid polypeptide and/or atherapeutically effective amount of at least one BKV-III capsidpolypeptide (or a fragment thereof) or a nucleic acid encoding the atleast one BKV-III capsid polypeptide. In some examples, the BKV-IIcapsid polypeptide (or fragment thereof) and the BKV-III capsidpolypeptide (or fragment thereof) are different from one another and arealso different from the BKV-I and BKV-IV capsid polypeptides (orfragments thereof). The BKV-II and BKV-III capsid polypeptides includeone or more of VP1, VP2, and VP3 (such as SEQ ID NOs: 7-12), and arediscussed in detail in Section IV, below. In particular examples,administering the at least one isolated BKV-II capsid polypeptideincludes administering a VLP including the BKV-II capsid polypeptide(s)and/or administering the at least one BKV-III capsid polypeptideincludes administering a VLP including the BKV-III capsidpolypeptide(s).

In some embodiments, the methods further include selecting a subject inneed of enhanced immunity to BKV. In some examples, a subject in need ofenhanced immunity to BKV is a subject at risk of BKV infection or atrisk of BKV-associated disorders, such as PVAN or BKV-associatedhemorrhagic cystitis. Subjects in need of enhanced immunity to BKVinclude subjects who are immunocompromised, for example subjects who areinfected with human immunodeficiency virus (HIV), subjects with SCID,diabetics, subjects who are receiving chemotherapy for cancer, andsubjects who are receiving immunosuppressive therapy (such ascorticosteroids, a calcineurin inhibitor, such as tacrolimus,cyclosporine, or pimecrolimus, or other therapies that decrease immunesystem function, such as rituximab, natalizumab, efalizumab, oralemtuzumab). In some examples, subjects who are receivingimmunosuppressive therapy include individuals who have received an organtransplant (such as a renal transplant or other solid organ transplantor a bone marrow transplant). In a particular example, a subject in needof enhanced immunity to BKV is a renal transplant recipient. In anotherexample, a subject in need of enhanced immunity to BKV is a bone marrowtransplant recipient. In other examples, subjects in need of enhancedimmunity to BKV include those who are candidates for organ or bonemarrow transplantation or those who are candidates for immunosuppressivetherapy. In a particular example, the subject has renal failure or isotherwise a candidate for a renal transplant. In further examples, asubject in need of enhanced immunity to BKV may include a subject whohas or is at risk for cancer (for example, prostate cancer or bladdercarcinoma).

In additional embodiments, the methods further include administering tothe subject a therapeutically effective amount of at least one JCVcapsid polypeptide (or a fragment thereof) or a nucleic acid encodingthe at least one JCV capsid polypeptide. The JCV capsid polypeptidesinclude one or more of VP1, VP2, and VP3 (for example, SEQ ID NOs:17-23), and are discussed in detail in Section IV, below. In particularexamples, administering the at least one isolated JCV capsid polypeptideincludes administering a VLP including the JCV capsid polypeptide(s).

In some examples, the subject who is administered the at least one JCVcapsid polypeptide or nucleic acid encoding the JCV capsid polypeptideis a subject in need of enhanced immunity to JCV or a subject at risk ofa JCV-associated disorder (for example, JCV-associated PML). Inparticular examples, the subject is an immunocompromised subject, forexample a subject who is infected with HIV, a subject with SCID, adiabetic subject, a subject who is receiving chemotherapy for cancer, ora subject who is receiving immunosuppressive therapy (such ascorticosteroids, a calcineurin inhibitor, such as tacrolimus,cyclosporine, or pimecrolimus, or other therapies that decrease immunesystem function, such as rituximab, natalizumab, efalizumab, oralemtuzumab). In some examples, a subject who is receivingimmunosuppressive therapy includes a subject who has received an organtransplant (such as a renal transplant or other solid organ transplantor a bone marrow transplant). In a particular example, a subject in needof enhanced immunity to JCV is a renal transplant recipient or a bonemarrow transplant recipient. In another example, a subject in need ofenhanced immunity to JCV is a subject who is receiving rituximab therapy(or a subject who will or has received rituximab therapy). In otherexamples, subjects in need of enhanced immunity to JCV include those whoare candidates for organ or bone marrow transplantation or those who arecandidates for immunosuppressive therapy.

B. Methods of Treating or Inhibiting Polyomavirus-Associated Disorders

In some embodiments, the methods include treating or inhibiting (or insome examples, even preventing) a polyomavirus-associated disorder, suchas PVAN, BKV-associated hemorrhagic cystitis, or JCV-associated PML. Insome examples, the methods include administering to a subject in need oftreatment for or inhibition of a polyomavirus-associated disorder atherapeutically effective amount of at least one isolated BKV-IV capsidpolypeptide (or a fragment thereof) or a nucleic acid encoding the atleast one BKV-IV capsid polypeptide to the selected subject (such as atleast one BKV-IVb1 VP1 polypeptide and/or at least one BKV-IVc2 VP1polypeptide). In some examples, administering the one or more BKV-IVcapsid polypeptides (such as VP1, VP2, or VP3, for example, SEQ ID NOs:4-6 and/or SEQ ID NO: 16) includes administering a VLP including thecapsid polypeptide(s).

In some embodiments, the methods further include selecting a subject inneed of treatment for inhibition of a polyomavirus-associated disorder.In some examples, a subject is need of treating or inhibiting PVAN orBK-associated hemorrhagic cystitis. (such as a subject who has had anorgan or bone marrow transplant or a candidate for an organ or bonemarrow transplant). In one example, the subject is a candidate for akidney transplant. In other examples, the subject is a candidate for abone marrow transplant. In further examples, the subject isimmunocompromised (such as a transplant recipient, a subject who isinfected with HIV, or a subject treated with an immunosuppressant), oris a candidate for treatment with an immunosuppressant (such as an organor bone marrow transplant candidate). In particular examples, thesubject does not have BKV-IV neutralizing antibodies and/or BKV-Ineutralizing antibodies.

In some examples, the methods further include administering to thesubject a therapeutically effective amount of at least one isolatedBKV-I capsid polypeptide (or a fragment thereof) or a nucleic acidencoding the at least one BKV-I capsid polypeptide, at least oneisolated BKV-II capsid polypeptide (or a fragment thereof) or a nucleicacid encoding the at least one BKV-II capsid polypeptide, at least oneisolated BKV-III capsid polypeptide (or a fragment thereof) or a nucleicacid encoding the at least one BKV-III capsid polypeptide, at least oneisolated JCV capsid polypeptide (or a fragment thereof) or a nucleicacid encoding the at least one JCV capsid polypeptide, or a combinationof two or more thereof (for example, one or more of SEQ ID NOs: 1-23).In particular non-limiting examples, the methods include administeringto the subject a therapeutically effective amount of at least one BKV-IaVP1 polypeptide and at least one BKV-Ib2 VP1 polypeptide (such as SEQ IDNOs: 1, 14, or 15). In some examples, administering the one or more BKVand/or JCV capsid polypeptides (such as VP1, VP2, or VP3) includesadministering a VLP including the capsid polypeptide(s).

In particular examples, the subject is a candidate for organ transplant(for example, renal transplant or bone marrow transplant) and thetherapeutically effective amount of the BKV-IV capsid polypeptide ornucleic acid encoding the capsid polypeptide is administered to thesubject a sufficient time prior to the organ transplant to produce animmune response to the BKV-IV capsid polypeptide in the subject. One ofskill in the art can identify the time required to produce an immuneresponse in the subject based on factors such as the general state ofthe subject's health, and the robustness of the subject's immune system.In some examples, the at least one isolated BKV-IV capsid polypeptide ornucleic acid encoding the BKV-IV capsid polypeptide is administered tothe subject at least about six months (for example, at least about 6months, 5 months, 4 months, 3 months, 2 months, 6 weeks, 5 weeks, 4weeks, 3 weeks, 2 weeks, or even 1 week) prior to the organ transplant.In some examples, the BKV-IV capsid polypeptide is administered to thesubject at least about 2 weeks prior to the organ transplant. In otherexamples, the BKV-IV capsid polypeptide is administered to the subjectat least about 6 weeks prior to the organ transplant, with a boosterdose about 2 weeks prior to transplant. In some examples, a BKV-I,BKV-II, BKV-III, and/or JCV capsid polypeptide (or fragment thereof) isfurther administered to the subject prior to the organ transplant.

In additional embodiments, the methods include administering to thesubject a therapeutically effective amount of a purified human gammaglobulin preparation that has been found to contain antibodies capableof neutralizing BKV-I, BKV-II, BKV-III and/or BKV-IV. Subjects includethose described above. Methods of identifying serum containing BKV-I,BKV-II, BKV-III, and/or BKV-IV neutralizing antibodies are known to oneof skill in the art and include the methods discussed in Section VI,below. In some examples, the gamma globulin preparations that may beused include commercially available preparations of intact gammaglobulin and preparations of the Fc, F(ab′) 2 fragments of gammaglobulin or combinations thereof. Methods of preparing gamma globulin,for example, for administration to a subject are known to one ofordinary skill in the art. See, e.g., U.S. Pat. Nos. 5,177,194;6,504,012; and 7,879,331.

The dosage of gamma globulin and the method of administration will varywith the severity and nature of the particular condition being treated,the duration of treatment, the adjunct therapy used, the age andphysical condition of the subject of treatment and similar factors. Insome examples, dosages for intravenous administration are from 100 mg/kgto 2.5 g/kg (such as about 400 mg/kg to 2 g/kg or 1 g/kg to 2 g/kg). Thedosage can be varied based on the frequency of administration, forexample, 400 mg/kg/day for 5 consecutive days per month or 2 g/kg/dayonce a month. In another example, the gamma globulin preparation isadministered subcutaneously or intramuscularly. In some examples, thedosage for subcutaneous or intramuscular administration is from about 1mg/kg to 30 mg/kg body weight (such as about 4 mg/kg to 20 mg/kg orabout 10 mg/kg to 20 mg/kg). The gamma globulin is administered as apharmaceutical composition containing a pharmaceutically acceptablecarrier. Pharmaceutically acceptable carriers of use for gamma globulincompositions include those described in section V, below.

C. Methods of Selecting a Renal Transplant Donor and/or Recipient

In further embodiments, the methods include selecting a candidate organtransplant donor and/or organ transplant recipient. In some examples,the candidate donor is a candidate renal transplant donor and thecandidate transplant recipient is a candidate renal transplantrecipient. The methods include screening a candidate donor and/orrecipient for presence of BKV serotype-specific antibodies (including,but not limited to BKV-IV-specific neutralizing antibodies).

In some embodiments, the methods include selecting a subject as a renaltransplant donor if BKV serotype-specific (such as BKV-IV or BKV-I)neutralizing antibodies are not present in the subject. In someexamples, the methods further include selecting a subject as a renaltransplant recipient if BKV serotype-specific (such as BKV-IV or BKV-I)neutralizing antibodies are not present in the subject who is acandidate transplant recipient. In some examples, the methods includedetecting presence of BKV-IV neutralizing antibodies in a subject (forexample, in a sample from a subject) and selecting the subject as atransplant donor or recipient if BKV-IV neutralizing antibodies are notpresent in the sample. The sample can include any suitable biologicalsample from the subject, including a blood sample or serum sample.

Methods of detecting neutralizing antibodies in a subject (such as in ablood or serum sample from a subject) are known to one of ordinary skillin the art. Such methods are discussed in Section VI, below.

IV. Polyomavirus Capsid Polypeptides

Polyomavirus nucleic acid and polypeptide sequences are publiclyavailable and can be identified by one of skill in the art. ExemplaryBKV genomic nucleic acid sequences include, but are not limited to,GenBank Accession Nos. JF894228, AB211374, DQ989796, AB211377, AB263920,AB211386, AB211390, and AB369093, each of which is incorporated hereinby reference as present in GenBank on Jul. 15, 2011.

It is disclosed herein that several BKV capsid polypeptides (orfragments thereof) can be used to elicit an immune response to BKV. Inseveral embodiments, the BKV capsid polypeptide comprises or consists ofthe amino acid sequence set forth as SEQ ID NOs: 1-12. Additional BKVVP1 polypeptides are disclosed herein, for example from additional BKVsubtypes. In some embodiments, the BKV VP1 polypeptide comprises orconsists of the amino acid sequence set forth as SEQ ID NOs: 13-16.

Exemplary JCV genomic nucleic acid sequences include, but are notlimited to, GenBank Accession Nos. NC_001699, AF300945, and AY536541,each of which is incorporated herein by reference as present in GenBankon Jul. 15, 2011.

It is also disclosed herein that several JCV capsid polypeptides (orfragments thereof) can be used to elicit an immune response to JCV, forexample, in combination with one or more BKV capsid polypeptides. Inseveral embodiments, the JCV capsid polypeptide comprises or consists ofthe amino acid sequence set forth as SEQ ID NOs: 17-23.

In some embodiments, the polyomavirus capsid polypeptides (such as BKVor JCV capsid polypeptides) of use in the methods disclosed herein havea sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%,such as 100% identical to the amino acid sequence set forth in one ofSEQ ID NOs: 1-23 or 52-125. In other examples, the polyomavirus capsidpolypeptides (such as BKV VP1 polypeptides) comprise a sequence at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, such as 100%identical to the amino acid sequence set forth in one of SEQ ID NOs:126-158. In some examples, the BKV subtype or serotype of thepolypeptide is known. In other examples, the BKV subtype or serotype ofthe polypeptide is not known. One of ordinary skill in the art candetermine the BKV subtype or serotype of an unknown BKV capsidpolypeptide, for example by sequence analysis and ELISA or neutralizingassays (such as those described in Examples 1-3, below). Exemplarysequences can be obtained using computer programs that are readilyavailable on the internet and the amino acid sequences set forth herein.In one example, the polypeptide retains a function of the polyomaviruscapsid polypeptide, such as binding to an antibody that specificallybinds the polyomavirus epitope.

Minor modifications of a polyomavirus capsid polypeptide primary aminoacid sequences may result in peptides which have substantiallyequivalent activity as compared to the unmodified counterpartpolypeptide described herein. Such modifications may be deliberate, asby site-directed mutagenesis, or may be spontaneous. All of thepolypeptides produced by these modifications are included herein. Thus,a specific, non-limiting example of a polyomavirus capsid polypeptide isa conservative variant of the polyomavirus capsid polypeptide (such as asingle conservative amino acid substitution, for example, one or moreconservative amino acid substitutions, for example 1-10 conservativesubstitutions, 2-5 conservative substitutions, 4-9 conservativesubstitutions, such as 1, 2, 5 or 10 conservative substitutions). Atable of conservative substitutions is provided herein. Substitutions ofthe amino acids sequence shown in SEQ ID NOs: 1-23 or 52-158 can be madebased on this table.

An “epitope” or “antigenic determinant” refers to a site on an antigento which B and/or T cells respond. T cells can respond to the epitopewhen the epitope is presented in conjunction with an MHC molecule.Epitopes can be formed both from contiguous amino acids (linear) ornoncontiguous amino acids juxtaposed by tertiary folding of an antigenicpolypeptide (conformational). Epitopes formed from contiguous aminoacids are typically retained on exposure to denaturing solvents whereasepitopes formed by tertiary folding are typically lost on treatment withdenaturing solvents. Normally, a B-cell epitope will include at leastabout 5 amino acids but can be as small as 3-4 amino acids. A T-cellepitope, such as a CTL epitope, will include at least about 7-9 aminoacids, and a helper T-cell epitope at least about 12-20 amino acids.Normally, an epitope will include between about 5 and 15 amino acids,such as 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids. In someexamples, the immunogenic compositions disclosed herein include afragment (such as an immunogenic fragment) or antigenic determinant of apolyomavirus capsid protein. One of skill in the art can identifypredicted antigenic determinants, for example using an HLA peptidebinding prediction program, such as BIMAS(www-bimas.cit.nih.gov/molbio/hla_bind/) or IEDB analysis resource(immuneeptiope.org). In some examples, the polyomavirus capsidpolypeptide includes, consists essentially of, or consists of five ormore amino acids (for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, or 23 amino acids) of the VP1 BC loop (e.g.,amino acids 61-83 of a BKV VP1 polypeptide; see, e.g., Tremolada et al.,Virus Res. 149:190-196, 2010).

The polyomavirus capsid polypeptides disclosed herein can be chemicallysynthesized by standard methods, or can be produced recombinantly. Anexemplary process for polypeptide production is described in Lu et al.,FEBS Lett. 429:31-35, 1998. They can also be isolated by methodsincluding preparative chromatography and immunological separations.Polypeptides can also be produced using molecular genetic techniques,such as by inserting a nucleic acid encoding at least one polyomaviruscapsid polypeptide or an epitope thereof into an expression vector,introducing the expression vector into a host cell, and isolating thepolypeptide. Any suitable cell can be utilized to express the disclosedpolypeptides, including bacteria (e.g., E. coli), yeast, insect cells(e.g., Sf9 cells), or mammalian cells (e.g., 293 cells). In someexamples, the polypeptide spontaneously assembles into a virus-likeparticle (VLP).

In some examples, the disclosed polyomavirus capsid polypeptides (orfragments thereof), for example a capsid polypeptide comprising theamino acid sequence of one or more of SEQ ID NOs: 1-23 or 52-125, are apart of a VLP, such as a BKV-I VLP, BKV-II VLP, BKV-III VLP, BKV-IV VLP,or JCV VLP. Immunogens are typically presented multimerically (e.g.,about 72 pentamers or about 360 capsid polypeptides per VLP particle) toimmune cells such as B cells and antigen presenting cells. This resultsin effectively inducing immune responses against the immunogen, inparticular, antibody responses. In some examples, the VLP includes oneor more of VP1, VP2, and VP3 (such as 1, 2, or all 3) from BKV-I (suchas BKV-Ia, BKV-Ib1, BKV-Ib2, and/or BKV-Ic), BKV-II, BKV-III, BKV-IV(such as BKV-IVb1 and/or BKV-IVc2), or JCV.

In specific embodiments, the antigen that is part of the disclosed VLPsincludes one or more of the amino acid sequences set forth as SEQ IDNOs: 1-23 or 52-158 (or fragments thereof) and have the ability tospontaneously assemble into VLPs. In some examples, a VLP includes aBKV-I VP1 polypeptide (such as one of SEQ ID NOs: 1, 13, 14, or 15) anda BKV-I VP2 polypeptide (such as SEQ ID NO: 2) and/or BKV-I VP3polypeptide (such as SEQ ID NO: 3). In other examples, a VLP includes aBKV-I VP1 polypeptide (such as one of SEQ ID NOs: 1, 13, 14, or 15) anda BKV-IV VP2 polypeptide (such as SEQ ID NO: 5) and/or BKV-IV VP3polypeptide (such as SEQ ID NO: 6). In other examples, a VLP includes aBKV-II VP1 polypeptide (such as SEQ ID NO: 7) and a BKV-II VP2polypeptide (such as SEQ ID NO: 8) and/or BKV-II VP3 polypeptide (suchas SEQ ID NO: 9). In other examples, a VLP includes a BKV-II VP1polypeptide (such as SEQ ID NO: 7) and a BKV-IV VP2 polypeptide (such asSEQ ID NO: 5) and/or BKV-IV VP3 polypeptide (such as SEQ ID NO: 6). Inadditional examples, a VLP includes a BKV-III VP1 polypeptide (such asSEQ ID NO: 10) and a BKV-III VP2 polypeptide (such as SEQ ID NO: 11)and/or BKV-III VP3 polypeptide (such as SEQ ID NO: 12). In otherexamples, a VLP includes a BKV-III VP1 polypeptide (such as SEQ ID NO:10) and a BKV-IV VP2 polypeptide (such as SEQ ID NO: 5) and/or BKV-IVVP3 polypeptide (such as SEQ ID NO: 6). In still further examples, a VLPincludes a BKV-IV VP1 polypeptide (such as one of SEQ ID NOs: 4 and 16)and a BKV-IV VP2 polypeptide (such as SEQ ID NO: 5) and/or BKV-IV VP3polypeptide (such as SEQ ID NO: 6). In another example, a VLP includes aJCV VP1 polypeptide (such as one of SEQ ID NOs: 17, 20, or 21) and a JCVVP2 polypeptide (such as SEQ ID NO: 22) and/or JCV VP3 polypeptide (suchas SEQ ID NO: 23).

In further examples, a fragment of a disclosed polyomavirus capsidpolypeptide retains the ability to spontaneously assemble into VLPs.Fragments (such as immunogenic fragments) and variants can be of varyinglength. For example, a fragment may consist of six or more, 25 or more,50 or more, 75 or more, 100 or more, or 200 or more amino acid residuesof a polyomavirus capsid amino acid sequence. This includes, forexample, any polypeptide six or more amino acid residues in length thatis capable of spontaneously assembling into VLPs. Methods to assay forVLP formation and isolation of VLPs are well known in the art (see, forexample, Pastrana et al., PLoS Pathogens 5(9):e1000578, 2009, hereinincorporated by reference in its entirety).

Polynucleotides encoding the BKV capsid polypeptides disclosed hereinare also provided. Exemplary nucleic acid sequences are set forth as SEQID NOs: 24-35. Polynucleotides encoding the JCV capsid polypeptidesdisclosed herein are also provided. Exemplary nucleic acid sequences areset forth as SEQ ID NOs: 36-38.

In some embodiments, the nucleic acids encoding the BKV capsidpolypeptides are codon-optimized for expression in a heterologous system(such as mammalian cells, bacteria or yeast). Exemplary nucleic acidsequences codon-optimized for mammalian cells are set forth as SEQ IDNOs: 39-51 (although the codon-optimized sequences can still beexpressed in other systems, such as bacteria).

In some embodiments, the nucleic acid sequences encoding polyomaviruscapsid polypeptides (such as BKV or JCV capsid polypeptides) of use inthe methods disclosed herein have a sequence at least 85%, 90%, 95%,96%, 97%, 98%, or 99%, such as 100% identical to the nucleic acidsequence set forth in one of SEQ ID NOs: 24-51. Exemplary sequences canbe obtained using computer programs that are readily available on theinternet and the nucleic acid sequences set forth herein. In oneexample, the polypeptide encoded by the nucleic acid sequence retains afunction of the polyomavirus capsid polypeptide, such as binding to anantibody that specifically binds the polyomavirus epitope.

V. Pharmaceutical Compositions and Modes of Administration

The polyomavirus capsid polypeptides (or fragments thereof) disclosedherein, or nucleic acids encoding the polyomavirus capsid polypeptides,can be used to elicit an immune response in a subject. In severalexamples, the subject is infected with at least one BKV serotype or isat risk of being infected with a BKV serotype (such as one or more ofBKV-I, BKV-II, BKV-III, or BKV-IV) and/or JCV. Thus, in severalembodiments, the methods include administering to a subject atherapeutically effective amount of one or more of the polyomaviruscapsid polypeptides (or fragments thereof) disclosed herein (orpolynucleotides encoding these polypeptides) in order to elicit animmune response, such as, but not limited to, a protective immuneresponse.

In the disclosed methods, compositions are administered to a subject inan amount sufficient to produce an immune response to a polyomavirus(for example, BKV). The disclosed BKV polypeptides, VLPs including theBKV polypeptides, or polynucleotides encoding these polypeptides, are ofuse to inhibit (or even prevent) an infection with BKV in a subject,inhibit (or even prevent) progression to disease in a subject having alatent BKV infection, or to inhibit or treat BKV-associated disorders(for example, PVAN or BKV-associated hemorrhagic cystitis) in a subjectinfected with BKV. In several examples, administration of atherapeutically effective amount of a composition including one or moreBKV serotype-specific capsid polypeptides (or fragments thereof)disclosed herein (or polynucleotides encoding these polypeptides)induces a sufficient immune response to decrease a symptom of a diseasedue to BKV infection, to inhibit the development of one or more symptomsof BKV or a BKV-associated disorder, or to inhibit infection with BKV(such as a BKV serotype, for example, BKV-I, BKV-II, BKV-III, and/orBKV-IV).

In some examples, the compositions are of use in inhibiting or evenpreventing a future infection with BKV. Thus, in some examples, atherapeutically effective amount of the composition is administered to asubject at risk of becoming infected with BKV (for example, animmunocompromised subject or a subject who has received or is acandidate for an organ transplant). The composition inhibits thedevelopment of BKV, such as latent or active BKV infection, in thesubject upon subsequent exposure to BKV, or loss of immunologicalcontrol over an existing BKV infection (for example reactivation of alatent infection).

In some embodiments, the methods further include administering to asubject a therapeutically effective amount of one or more of the JCVcapsid polypeptides (or fragment thereof) disclosed herein (orpolynucleotides encoding these polypeptides) in order to elicit animmune response, such as, but not limited to, a protective immuneresponse against JCV. In some examples, the compositions are of use ininhibiting or even preventing a future infection with JCV. Thus, in someexamples, a therapeutically effective amount of the composition isadministered to a subject at risk of becoming infected with JCV (forexample, an immunocompromised subject or a subject who has or is acandidate for organ transplantation). The composition inhibits orprevents the development of JCV, such as latent or active JCV infection,in the subject upon subsequent exposure to JCV, or loss of immunologicalcontrol over an existing JCV infection (for example reactivation of alatent infection). In some examples, the disclosed methods andcompositions inhibit or treat JCV-associated disorders (for example,JCV-associated PML) in a subject infected with JCV. In particularexamples, the subject is at risk of developing JCV-associated PML, suchas a subject infected with HIV, a subject on immune-suppressing therapy(for example, mycophenolate, fludarabine, methotrexate, rituximab,natalizumab, alemtuzumab, or efalizumab), or a subject who has, or is acandidate for, organ transplantation (such as a bone marrow transplant).

Amounts effective for these uses will depend upon the severity of thedisease, the general state of the subject's health, and the robustnessof the subject's immune system. In one example, a therapeuticallyeffective amount of the compound is that which provides eithersubjective relief of a symptom or an objectively identifiableimprovement as noted by the clinician or other qualified observer. Inother examples, a therapeutically effective amount is an amountsufficient to inhibit an infection with BKV (such as BKV-I, BKV-II,BKV-III, and/or BKV-IV) in a subject upon subsequent exposure of thesubject to one or more BKV serotypes. In additional examples, atherapeutically effective amount is an amount sufficient to inhibitdevelopment of one or more symptoms in a subject infected with BKV (forexample, PVAN or BKV-associated hemorrhagic cystitis).

In further examples, a therapeutically effective amount is an amountsufficient to inhibit an infection with JCV in a subject upon subsequentexposure of the subject to one or more JCV serotypes or inhibit theemergence of an existing JCV infection from asymptomatic latency in asubject. In additional examples, a therapeutically effective amount isan amount sufficient to inhibit development of one or more symptoms in asubject infected with JCV (for example, JCV-associated PML).

In some examples, one or more polyomavirus capsid polypeptides (such asBKV or JCV capsid polypeptides) or fragments thereof described hereinmay be covalently linked to at least one other immunogenic protein,wherein the conjugate elicits an immune response to the polyomaviruscapsid polypeptide in a subject. The other immunogenic protein(sometimes referred to as a “carrier” protein) ideally has theproperties of being immunogenic by itself, usable in a subject, and of asize that can be easily purified and conjugated to at least one otherprotein or peptide. Suitable carrier proteins are known to one of skillin the art. In particular examples, the other immunogenic protein(carrier protein) is bovine serum albumin (BSA), ovalbumin, tetanustoxoid, diphtheria toxoid, cholera toxin, Clostridium difficile toxin A,C. difficile toxin B, Shiga toxin, or Pseudomonas aeruginosa recombinantexoprotein A.

A polyomavirus capsid polypeptide can be administered by any means knownto one of skill in the art (see Banga, A., “Parenteral ControlledDelivery of Therapeutic Peptides and Proteins,” in Therapeutic Peptidesand Proteins, Technomic Publishing Co., Inc., Lancaster, Pa., 1995)either locally or systemically, such as by intramuscular injection,subcutaneous injection, intraperitoneal injection, intravenousinjection, oral administration, nasal administration, transdermaladministration, or even anal administration. In some embodiments,administration is by oral administration, subcutaneous injection, orintramuscular injection.

In one specific, non-limiting example, the polyomavirus capsidpolypeptide is administered in a manner to direct the immune response toa cellular response (that is, a helper T cell or cytotoxic T lymphocyte(CTL) response), rather than a humoral (antibody) response.

To extend the time during which the peptide or protein is available tostimulate a response, the peptide or protein can be provided as animplant, an oily injection, or as a particulate system. The particulatesystem can be a microparticle, a microcapsule, a microsphere, ananocapsule, or similar particle. (see, e.g., Banga, supra). Aparticulate carrier based on a synthetic polymer has been shown to actas an adjuvant to enhance the immune response, in addition to providinga controlled release. Aluminum salts can also be used as adjuvants toproduce an immune response.

Optionally, one or more cytokines, such as IL-2, IL-6, IL-12, RANTES,GM-CSF, TNF-α, or IFN-γ, one or more growth factors, such as GM-CSF orG-CSF; one or more molecules such as OX-40L or 4-1 BBL, or combinationsof these molecules, can be used as biological adjuvants (see, forexample, Salgaller et al., 1998, J. Surg. Oncol. 68(2):122-38; Lotze etal., 2000, Cancer J. Sci. Am. 6(Suppl 1):S61-6; Cao et al., 1998, StemCells 16(Suppl 1):251-60; Kuiper et al., 2000, Adv. Exp. Med. Biol.465:381-90). These molecules can be administered systemically (orlocally) to the host. In several examples, IL-2, RANTES, GM-CSF, TNF-α,IFN-γ, G-CSF, LFA-3, CD72, B7-1, B7-2, B7-1 B7-2, OX-40L, 4-1 BBL,and/or ICAM-1 are administered.

A pharmaceutical composition including one or more polyomavirus capsidpolypeptide is thus provided. These compositions are of use to promotean immune response to polyomavirus, such as a BKV serotype-specificresponse. In some examples, the disclosed compositions include a BKV-IVcapsid polypeptide (or a fragment thereof), a BKV-I capsid polypeptide(or a fragment thereof), and a pharmaceutically acceptable carrier. Inparticular examples, the compositions include a VLP including at leastone BKV-IV capsid polypeptide, a VLP including at least one BKV-I capsidpolypeptide, and a pharmaceutically acceptable carrier. In otherexamples, the composition includes a VLP including at least one BKV-Iacapsid polypeptide, a VLP including at least one BKV-Ib2 capsidpolypeptide, a VLP including at least one BKV-IV capsid polypeptide, anda pharmaceutically acceptable carrier. In some examples, thecompositions further include at least one BKV-II capsid polypeptide, atleast one BKV-III capsid polypeptide and/or at least one JCV capsidpolypeptide (such as one or more VLPs including at least one BKV-IIcapsid polypeptide, at least one BKV-III capsid polypeptide, and/or atleast one JCV capsid polypeptide). In some embodiments, the compositionsinclude one or more adjuvants.

In one embodiment, the polyomavirus capsid polypeptide is mixed with anadjuvant containing two or more of a stabilizing detergent, amicelle-forming agent, and an oil. Suitable stabilizing detergents,micelle-forming agents, and oils are detailed in U.S. Pat. Nos.5,585,103; 5,709,860; 5,270,202; and 5,695,770, all of which areincorporated by reference. A stabilizing detergent is any detergent thatallows the components of the emulsion to remain as a stable emulsion.Such detergents include polysorbate, 80 (TWEEN)(Sorbitan-mono-9-octadecenoate-poly(oxy-1,2-ethanediyl; manufactured byICI Americas, Wilmington, Del.), TWEEN 40™, TWEEN 20™, TWEEN 60™,ZWITTERGENT™ 3-12, TEEPOL HB7™, and SPAN 85™. These detergents areusually provided in an amount of approximately 0.05 to 0.5%, such as atabout 0.2%. A micelle forming agent is an agent which is able tostabilize the emulsion formed with the other components such that amicelle-like structure is formed. Such agents generally cause someirritation at the site of injection in order to recruit macrophages toenhance the cellular response. Examples of such agents include polymersurfactants described by BASF Wyandotte publications, e.g., Schmolka, J.Am. Oil. Chem. Soc. 54:110, 1977; and Hunter et al., J. Immunol.127:1244-1250, 1981; for example, PLURONIC™ L62LF, L101, and L64,PEG1000, and TETRONIC™ 1501, 150R1, 701, 901, 1301, and 130R1. Thechemical structures of such agents are well known in the art. In oneembodiment, the agent is chosen to have a hydrophile-lipophile balance(HLB) of between 0 and 2, as defined by Hunter and Bennett, J. Immunol.133:3167-3175, 1984. The agent can be provided in an effective amount,for example between 0.5 and 10%, or in an amount between 1.25 and 5%.

The oil included in the composition is chosen to promote the retentionof the antigen in oil-in-water emulsion, such as to provide a vehiclefor the desired antigen, and preferably has a melting temperature ofless than 65° C. such that emulsion is formed either at room temperature(about 20° C. to 25° C.), or once the temperature of the emulsion isbrought down to room temperature. Examples of such oils includesqualene, squalane, EICOSANE™, tetratetracontane, glycerol, and peanutoil or other vegetable oils. In one specific, non-limiting example, theoil is provided in an amount between 1 and 10%, or between 2.5 and 5%.The oil should be both biodegradable and biocompatible so that the bodycan break down the oil over time, and so that no adverse affects, suchas granulomas, are evident upon use of the oil.

In one embodiment, the adjuvant is a mixture of stabilizing detergents,micelle-forming agent, and oil available under the name PROVAX® (IDECPharmaceuticals, San Diego, Calif.). An adjuvant can also be animmunostimulatory nucleic acid, such as a nucleic acid including a CpGmotif, or a biological adjuvant (see above).

Controlled release parenteral formulations can be made as implants, oilyinjections, or as particulate systems. For a broad overview of proteindelivery systems, see Banga, Therapeutic Peptides and Proteins:Formulation, Processing, and Delivery Systems, Technomic PublishingCompany, Inc., Lancaster, Pa., 1995. Particulate systems includemicrospheres, microparticles, microcapsules, nanocapsules, nanospheres,and nanoparticles. Microcapsules contain the therapeutic protein as acentral core. In microspheres, the therapeutic agent is dispersedthroughout the particle. Particles, microspheres, and microcapsulessmaller than about 1 μm are generally referred to as nanoparticles,nanospheres, and nanocapsules, respectively. Capillaries have a diameterof approximately 5 μm so that only nanoparticles are administeredintravenously. Microparticles are typically around 100 μm in diameterand are administered subcutaneously or intramuscularly (see Kreuter,Colloidal Drug Delivery Systems, J. Kreuter, ed., Marcel Dekker, Inc.,New York, N.Y., pp. 219-342, 1994; Tice & Tabibi, Treatise on ControlledDrug Delivery, A. Kydonieus, ed., Marcel Dekker, Inc. New York, N.Y.,pp. 315-339, 1992).

Polymers can be used for controlled release. Various degradable andnondegradable polymeric matrices for use in controlled drug delivery areknown in the art (Langer, Accounts Chem. Res. 26:537, 1993). Forexample, the block copolymer, polaxamer 407 exists as a viscous yetmobile liquid at low temperatures but forms a semisolid gel at bodytemperature. It has shown to be an effective vehicle for formulation andsustained delivery of recombinant interleukin-2 and urease (Johnston etal., Pharm. Res. 9:425, 1992; and Pec, J. Parent. Sci. Tech. 44(2):58,1990). Alternatively, hydroxyapatite has been used as a microcarrier forcontrolled release of proteins (Ijntema et al., Int. J. Pharm. 112:215,1994). In yet another aspect, liposomes are used for controlled releaseas well as drug targeting of the lipid-capsulated drug (Betageri et al.,Liposome Drug Delivery Systems, Technomic Publishing Co., Inc.,Lancaster, Pa., 1993). Numerous additional systems for controlleddelivery of therapeutic proteins are known (e.g., U.S. Pat. Nos.5,055,303; 5,188,837; 4,235,871; 4,501,728; 4,837,028; 4,957,735;5,019,369; 5,055,303; 5,514,670; 5,413,797; 5,268,164; 5,004,697;4,902,505; 5,506,206; 5,271,961; 5,254,342; and 5,534,496).

In another embodiment, a pharmaceutical composition includes a nucleicacid encoding a polyomavirus capsid polypeptide or fragment thereof (forexample, a BKV or JCV capsid polypeptide or fragment). A therapeuticallyeffective amount of the BKV or JCV capsid polynucleotide can beadministered to a subject in order to generate an immune response.

One approach to administration of nucleic acids is direct immunizationwith plasmid DNA, such as with a mammalian expression plasmid. Forexample, the nucleotide sequence encoding a polyomavirus capsidpolypeptide can be placed under the control of a promoter to increaseexpression of the molecule.

Immunization by nucleic acid constructs is well known in the art andtaught, for example, in U.S. Pat. No. 5,643,578 (which describes methodsof immunizing vertebrates by introducing DNA encoding a desired antigento elicit a cell-mediated or a humoral response), and U.S. Pat. Nos.5,593,972 and 5,817,637 (which describe operably linking a nucleic acidsequence encoding an antigen to regulatory sequences enablingexpression). U.S. Pat. No. 5,880,103 describes several methods ofdelivery of nucleic acids encoding immunogenic peptides or otherantigens to an organism. The methods include liposomal delivery of thenucleic acids (or of the synthetic peptides themselves), andimmune-stimulating constructs, or ISCOMS™ (negatively charged cage-likestructures of 30-40 nm in size formed spontaneously on mixingcholesterol and saponin). Protective immunity has been generated in avariety of experimental models of infection, including toxoplasmosis andEpstein-Barr virus-induced tumors, using ISCOMS™ as the delivery vehiclefor antigens (Mowat and Donachie, Immunol. Today 12:383, 1991). Doses ofantigen as low as 1 μg encapsulated in ISCOMS™ have been found toproduce Class I mediated CTL responses (Takahashi et al., Nature344:873, 1990).

Optionally, one or more cytokines, such as IL-2, IL-6, IL-12, RANTES,GM-CSF, TNF-α, or IFN-γ, one or more growth factors, such as GM-CSF orG-CSF, one or more costimulatory molecules, such as ICAM-1, LFA-3, CD72,B7-1, B7-2, or other B7 related molecules; one or more molecules such asOX-40L or 4-1 BBL, or combinations of these molecules, can be used asbiological adjuvants (see, for example, Salgaller et al., 1998, J. Surg.Oncol. 68(2):122-38; Lotze et al., 2000, Cancer J. Sci. Am. 6(Suppl1):S61-6; Cao et al., 1998, Stem Cells 16(Suppl 1):251-60; Kuiper etal., 2000, Adv. Exp. Med. Biol. 465:381-90). These molecules can beadministered systemically to the host. It should be noted that thesemolecules can be co-administered via insertion of a nucleic acidencoding the molecules into a vector, for example, a recombinant poxvector (see, for example, U.S. Pat. No. 6,045,802). In variousembodiments, the nucleic acid encoding the biological adjuvant can becloned into same vector as the BKV polypeptide coding sequence, or thenucleic acid can be cloned into one or more separate vectors forco-administration.

In one embodiment, a nucleic acid encoding a polyomavirus capsidpolypeptide is introduced directly into cells. For example, the nucleicacid can be loaded onto gold microspheres by standard methods andintroduced into the skin by a device such as the Helios™ Gene Gun(Bio-Rad, Hercules, Calif.). The nucleic acids can be “naked,”consisting of plasmids under control of a strong promoter. Typically,the DNA is injected into muscle, although it can also be injecteddirectly into other sites. Dosages for injection are usually around 0.5μg/kg to about 50 mg/kg, and typically are about 0.005 mg/kg to about 5mg/kg (see, for example, U.S. Pat. No. 5,589,466).

In one specific, non-limiting example, a pharmaceutical composition forintravenous administration would include about 0.1 μg to 100 mg ofimmunogenic polyomavirus capsid polypeptide (or fragment thereof) perpatient per day. Dosages from 0.1 to about 100 mg per patient per day(for example, about 10 mg to 50 mg) can be used, particularly if theagent is administered to a secluded site and not into the circulatory orlymph system, such as into a body cavity or into a lumen of an organ. Inother non-limiting examples, the pharmaceutical composition includes oneor more VLPs including the disclosed polyomavirus capsid polypeptides,for example about 1-200 μg VLP (such as about 10 μg to 200 μg, about 20μg to 100 μg, or about 20 μg to about 40 μg).

In some examples, the compositions include pharmaceutically acceptablecarriers and/or one or more adjuvants. Actual methods for preparingadministrable compositions will be known or apparent to those skilled inthe art and are described in more detail in such publications asRemington: The Science and Practice of Pharmacy, The University of theSciences in Philadelphia, Editor, Lippincott, Williams, & Wilkins,Philadelphia, Pa., 21^(st) Edition (2005).

The administration of the polyomavirus capsid polypeptides (or fragmentsthereof), VLPs including the polypeptides, or nucleic acids encoding thepolypeptides can be sequential, simultaneous (concurrent), orsubstantially simultaneous. Sequential administration can be separatedby any amount of time, so long as the desired effect is achieved. Insome examples a BKV-I capsid polypeptide or fragment thereof and aBKV-IV capsid polypeptide or fragment thereof are administered to asubject sequentially. In other examples, a BKV-I capsid polypeptide orfragment thereof and a BKV-IV capsid polypeptide or fragment thereof areadministered to a subject simultaneously or substantiallysimultaneously.

In some examples, the effectiveness of the therapeutic or preventiveintervention is monitored by titering the BKV or JCV neutralizingpotential of the subject's serum antibody responses over time. Subjectswho are found to have been poorly responsive to initial therapeuticinterventions, such as but not limited to immunization with BKV or JCVcapsid proteins, are given one or more booster doses of the therapeuticintervention.

Multiple administrations of the compositions described herein are alsocontemplated. Single or multiple administrations of the compositions areadministered, depending on the dosage and frequency as required andtolerated by the subject. In one embodiment, the dosage is administeredonce as a bolus, but in another embodiment can be applied periodicallyuntil a therapeutic result is achieved. In one embodiment, the dose issufficient to treat or ameliorate symptoms or signs of BKV and/or JCVwithout producing unacceptable toxicity to the subject. In anotherembodiment, the dose is sufficient to inhibit infection with BKV uponsubsequent exposure to BKV. In other embodiments, the dose is sufficientto inhibit infection with JCV upon subsequent exposure to JCV. In afurther embodiment, the dose is sufficient to inhibit a symptom of BKVin a subject with a latent BKV infection. In another embodiment, thedose is sufficient to inhibit a symptom of JCV in a subject with alatent JCV infection. Systemic or local administration can be utilized.

VI. Methods of Monitoring Immune Response to Polyomavirus

Also disclosed herein are methods of monitoring an immune response topolyomavirus, for example, following exposure (or potential exposure) topolyomavirus or following immunization against polyomavirus. In someexamples, the methods include detecting the presence of polyomavirusantibodies in a subject that has been administered an immunogeniccomposition comprising at least one isolated BKV capsid polypeptide (orfragment thereof) or a nucleic acid encoding a BKV capsid polypeptide.In other examples, the methods include detecting the presence ofpolyomavirus antibodies in a subject that has been administered animmunogenic composition comprising at least one isolated JCV capsidpolypeptide (or fragment thereof) or a nucleic acid encoding a JCVcapsid polypeptide.

In some examples, the method includes detecting the presence ofpolyomavirus antibodies (such as neutralizing antibodies) in a samplefrom a subject (such as a blood sample or serum sample). Thepolyomavirus antibodies include one or more of BKV-I antibodies (such asBKV-Ia antibodies, BKV-Ib2 antibodies, and/or BKV-Ic antibodies), BKV-IIantibodies, BKV-III antibodies, BKV-IV antibodies (such as BKV-IVb1antibodies and/or BKV-IVc2 antibodies), or JCV antibodies. In someexamples, the antibodies are neutralizing antibodies. In somenon-limiting examples, the antibodies are BKV-Ia neutralizingantibodies, BKV-Ib2 neutralizing antibodies, or BKV-IV neutralizingantibodies. Monitoring immune response to polyomavirus can indicatewhether a subject has developed an immune response (for example, aprotective immune response) to one or more polyomaviruses, for example,following administration of an immunogenic composition (for example, asdescribed above). Monitoring immune response to polyomavirus can alsoindicate whether a subject has seroconverted for one or morepolyomaviruses (for example, BKV-I and/or BKV-IV) following an organtransplant or immunosuppressive therapy. In some examples, multiplesamples from a subject are tested for presence of antibodies over time,for example prior to and at time points after (such as 1 week, 2 weeks,1 month, 2 months, 3 months, 6 months, 9 months, 1 year, or more after)administration of an immunogenic composition, organ transplantation orstart of immunosuppressive therapy.

Methods for detecting antibodies in a sample are known to one of skillin the art. Such methods include but are not limited to ELISA,immunofluorescence assay, radioimmunoassay, and micro-agglutinationtest. In some examples, the methods include detecting the presence ofneutralizing antibodies (such as BKV serotype-specific neutralizingantibodies) in a sample from a subject. In some examples, assays fordetecting neutralizing antibodies include plaque reductionneutralization test, cell killing, and reporter assays.

In a particular example, neutralizing antibodies are detected using areporter assay. BKV or JCV reporter vectors (also known aspseudovirions) are produced by packaging a reporter plasmid in cells(such as 293 cells, for example 293TT cells or 293FT cells for BKV orSVG cells for JCV) expressing a BKV (such as BKV-I, BKV-II, BKV-III, orBKV-IV) or JCV capsid polypeptide (for example, VP1, VP2, and/or VP3).The reporter vector particles are then isolated and treated with serialdilutions of serum from a subject (such as a series of four-folddilutions from 1:100 to 1:2.6×10⁷ or a series of 10-fold dilutions from1:100 to 1×10⁷). The serum/reporter vector mixture is then applied tofresh cells (for example 293 cells for BKV or SVG cells for JCV) for aperiod of time (such as 72 hours). The cell culture is then assayed forproduction of a reporter protein encoded by the reporter plasmidpackaged within the reporter vector. A decrease in reporter vectoractivity (for example, as compared to a control, such as a no serumcontrol) indicates the presence of neutralizing antibodies in thesample. In one example, the reporter plasmid carries an SV40 origin ofreplication, which can mediate replicative amplification of thetransduced plasmid in the target cell. In a specific example, thereporter vector is phGluc (Gaussia princeps luciferase under the controlof a human elongation factor 1 alpha promoter) and activity is detectedusing Gaussia luciferase substrate (New England Biolabs). See, e.g.,Pastrana et al., PLoS Pathogens 5(9):e1000578, 2009 (incorporated hereinby reference). One of ordinary skill in the art can select additionalreporters of use in neutralizing antibody assays, such as greenfluorescent protein, β-galactosidase, alkaline phosphatase, and others.

The present disclosure is illustrated by the following non-limitingExamples.

Example 1 Materials and Methods

Mice: Eight-week old female BALB/cAnNCr mice were immunized oncesubcutaneously with 5 μg of BKV-I or BKV-IV viral-like particles (VLPs)in complete Freund's Adjuvant (CFA, Sigma-Aldrich, St. Louis, Mo.). Serawere obtained 4 weeks after immunization. The animals were kept underpathogen-free conditions in compliance with institutional guidelines atthe National Cancer Institute.

Sera: Samples from 108 renal transplant subjects from the “RandomizedProspective Controlled Clinical and Pharmacoeconomic Study ofCyclosporine vs. Tacrolimus in Adult Renal Transplant Recipients” of theWashington University, School of Medicine were used. The patients andclinical protocols from the study have previously been described indetail (Bohl et al., Am. J. Transplant. 5:2213-2221; Randhawa et al.,Clin. Vaccine Immunol. 15:1564-1571, 2008). Patients were given animmunosuppressive regimen, which was discontinued if viremia wasdetected. Serum samples were collected at roughly 1, 4, 12, 26 and 52weeks post-transplantation. None of the patients were observed to sufferfrom PVAN during the course of the collection period. Sera from healthysubjects visiting U.S. plasma donation centers have been described indetail before (Pastrana et al., PLoS Pathog. 5:e1000578, 2009).

VLPs and reporter vectors (pseudovirions): BKV reporter vectors weregenerated as previously described (Pastrana et al., PLoS Pathog.5:e1000578, 2009). BKV-I reporter vectors were produced using plasmidpCAG-BKV (Nakanishi et al., Virology 379:110-117, 2008), which encodesthe capsid proteins of BKV isolate KOM-5 (SEQ ID NOs: 1-3). KOM-5 isclassified as a BKV type I subtype b-1 (Ib-1) genotype (Nishimoto etal., J. Mol. Evol. 63:341-352, 2006), and was transfected into 293TTcells (Buck et al., J. Virol. 78:751-757, 2004) using Lipofectamine 2000(Invitrogen, Carlsbad, Calif.). Then, 48 hours after transfection thecells were suspended at >100 million cells/ml in Dulbecco's phosphatebuffered saline (DPBS) and lysed by addition of 0.5% Triton X-100, 25 mMammonium sulfate (diluted from a 1M stock adjusted to pH 9) and RNaseA/T1cocktail (Ambion, Austin, Tex.). The lysate was incubated at 37° C.overnight to allow capsid maturation, then clarified by spinning twicefor 10 minutes at 5,000×g, with gentle agitation of the lysate betweenspins. Reporter vector particles were purified out of the clarifiedsupernatant through a 27-33-39% iodixanol (OptiPrep®, Sigma-Aldrich, St.Louis, Mo.) step gradient (Buck and Thompson, Curr. Protoc. Cell Biol.Chapter 26, Unit 26.21, 2007).

For BKV-IV particles the sequence of BKV isolate A-66H (subtype IV-c2;Zhong et al., J. Gen. Virol. 90:144-152, 2009) was used to designsynthetic codon-modified versions of the VP1, VP2 and VP3 genes (SEQ IDNOs: 39-41). The codon-modified genes were synthesized by Blue HeronBiotechnology (Bothell, Wash.) and cloned into Gateway (Invitrogen)adapted mammalian expression plasmids pGwf (for VP1) or phGf (for VP2and VP3) (Buck et al., Proc. Natl. Acad. Sci. USA 103:1516-1521, 2006).An additional pair of plasmids, pwB2b and pwB3b, were generated bytransferring the BKV-IV VP2 or VP3 (respectively) gene into the SV40promoter-driven expression cassette of pwB. For BKV-IV reporter vectorproduction, cells were co-transfected with pwB2b, pwB3b, ph2b, ph3b andphGluc at a 2:2:1:1:1 ratio. Initial particle stocks produced withoutph2b or ph3b in the co-transfection mixture appeared to show poor VP2/3occupancy and relatively poor infectivity. Transfection, harvesting andpurification of BKV-IV reporter vectors were the same as for productionof BKV-I reporter vectors.

For generation of VLPs, 293TT cells were transfected with pCAG-BKV(BKV-I) or a mixture of pwB2b and pwB3b (BKV-IV) without any reporterplasmid. Two days after transfection, the cells were lysed with 0.5%Triton X-100 in DPBS supplemented with 25 mM ammonium sulfate, Benzonase(Sigma-Aldrich), Plasmid Safe (Epicentre, Madison, Wis.), and 1.2 U/mlneuraminidase V (Sigma-Aldrich, Catalog No. N2876). The lysates wereincubated at 37° C. overnight, then adjusted to 0.85 M NaCl, clarifiedas above, and subjected to purification over OptiPrep® gradients.

ELISAs: Immulon™ H2B plates (Thermo Fisher Scientific, Waltham, Mass.)were coated with 15 ng/well of VLPs in PBS overnight. PBS with 1%non-fat dry milk (blotto) was used to block the coated plates for 2hours at room temperature, with orbital rotation. Sera from mice andhealthy human subjects were serially diluted in blotto and incubated onblocked plates at room temperature for 1 hour, with orbital rotation.Washing was performed with PBS. Horseradish peroxidase conjugated goatanti-mouse IgG (BioRad) or donkey anti-human IgG (JacksonImmunoResearch, Wes Grove, Pa.) diluted 1:7500 in blotto was used todetect bound sera. The plates were incubated with ABTS(2,2-azino-di43-ethylbenzthiazoine sulfonate) substrate (Roche AppliedScience, Indianapolis, Ind.) and absorbance read at 405 nm with areference read at 490 nm. The effective concentration 10% (EC₁₀) wascalculated using Prism® software (GraphPad Software, La Jolla, Calif.)to fit a curve to the OD values for each serially-diluted serum sample.The top of each response curve was constrained based on the average ofthe calculated plateau maximum (Bmax) values for strongly reactive sera.The Bmax value was typically an OD value of around 2.0, such that theEC₁₀ value can be considered comparable to an OD cutoff value of 0.2.

Neutralization Assays: Neutralization assays were performed aspreviously reported (Pastrana et al., PLoS Pathog. 5:e1000578, 2009).Briefly, 293TT cells were seeded at a density of 3×10⁴ cells per welland allowed to attach for 3-5 hours. Sera from mice and human subjectswere serially diluted, and sera from renal transplant patients weretested in separate assays at 4 different dilutions: 1:100, 1:500,1:5,000, and 1:50,000. Dilutions were performed in cell culture medium(DMEM without phenol red supplemented with 25 mM HEPES, 10%heat-inactivated fetal bovine serum, 1% MEM non-essential amino acids,1% Glutamax™ supplement and 1% antibiotic-antimycotic, all fromInvitrogen). Then 24 μl of diluted sera were mixed with 96 μl of dilutedreporter vector stock and placed at 4° C. for 1 hour. Cells wereincubated with 100 μl of this mixture for 72 hours. Conditionedsupernatants (25 μl) were harvested into white 96-well luminometryplates (Perkin Elmer, Waltham, Mass.). Gaussia Luciferase Assay Kitsubstrate (50 New England Biolabs, Ipswich, Mass.) was injectedimmediately prior to luminometry using a BMG Labtech Polarstar Optimaluminometer.

For mice and sera from healthy individuals, 50% neutralizing titers(EC₅₀) were calculated based on dose-response curves with top and bottomvalues constrained to the average values of “no serum” and “no reportervector” control wells, respectively. For transplant patients, thefollowing criteria for seropositivity and seronegativity were adopted:sera were considered negative at entry if the 1:100 dilution did notmediate at least a 95% reduction in Gaussia luciferase activity(measured in relative light units, RLUs) relative to the no serumcontrol condition (>95% neutralization of the reporter vector).Seroconversion refers to subjects who scored seronegative at the initialtime point but whose sera were >95% neutralizing at the 1:500 dilutionat any subsequent time point. A stricter definition of seroconversion,accounting for the possible low level cross-type neutralization, addedthe stipulation that the 95% neutralizing titer for BKV-IV differed fromthe BKV-I neutralizing titer by less than 1,000 fold.

Sequence analysis: VP1 sequences from BKV strains indicated in Table 3(below) were downloaded from GenBank (Pastrana et al., PLoS Pathogens8:e1002650, 2012; incorporated herein by reference in its entirety).ClustalW alignments were performed with MacVector software version11.1.2 using a Gonnet series matrix. Structural modeling of BKV VP1amino acid variations was performed by aligning the sequences of JCV orSV40 VP1 to BKV, followed by inspection of homologous positions ofinterest in the JCV or SV40 VP1 X-ray crystal structures (PDB IDaccession numbers 3NXD and 1SVA, respectively). Structure inspectionswere performed using Swiss PDB Viewer.

Example 2 BKV Cross-Neutralizing Responses in Mouse Sera

This example describes serological cross-reactivity in mice immunizedwith VLPs from BKV-I or BKV-IV serotypes.

To determine if reactivity to one BKV type would generate cross-reactiveantibodies, mice were immunized with VLPs containing the VP1, VP2, andVP3 capsid proteins from the two most common serotypes: BKV-I (BKV-Ib1isolate KOM-5) or BKV-IV (BKV-IVc2 isolate A-66H). A singlesub-cutaneous dose in the presence of Freund's adjuvant resulted in thedevelopment of high-titer responses. As measured by ELISA, all mice butone responded with titers against BKV-I ranging from 9000 to 110,000(FIG. 1 , top panel). The response in BKV-IV immunized mice was similar,with titers ranging from 13,000 to 130,000. The sera exhibited varyingamounts of cross-reactivity against the non-cognate BKV. The averageratio of homologous to heterologous titer was 21 for mice immunized withBKV-I and 110 for mice immunized with BKV-IV (FIG. 1 , bottom panel).For this calculation the non-responsive mouse was eliminated, as thedenominator was not a true titer, but arbitrarily set at 25, or thelowest concentration tested.

In order to obtain more information on the cross-neutralizing responses,a reporter-vector based neutralizing assay was also utilized. Theserecombinant production systems made it possible to generate infectiouscapsids composed of the VP1/2/3 capsid proteins of BKV primary isolatesof genotypes I and IV that are not otherwise culturable. Using thereporter-based assays, the neutralizing potency of sera from BKV-I orBKV-IV vaccinated mice were titered. The neutralizing assay BKV-I, whencompared to ELISAs, has been shown to have a broader linear range fordetection of serum titers (Pastrana et al., PLoS Pathog. 5:e1000578,2009), and a similar neutralization assay has also shown improvedspecificity in the context of papillomaviruses (Pastrana et al.,Virology 321:205-216, 2004). The neutralization assays showed asignificantly greater degree of BKV type-specificity compared to theELISAs. For BKV-I immunized mice, homologous titers ranged from 1100 to3,000,000 (FIG. 1 , middle panel). The mouse that was non-reactive byELISA showed a titer, of 1100, which was 55-fold lower than the nextmouse with lower BKV-I titers. Mice immunized with BKV-IV had titersranging from 17,000 to 600,000. The ratio of homologous to heterologoustiter was 910 for mice immunized with BKV-I and 620 for mice immunizedwith BKV-IV. A comparison of ELISA values to neutralization assay valuesis shown in FIG. 2 .

To test the possibility that a booster vaccination might alter thedegree of cross-neutralization of the two BKV types, a second dose ofcognate VLPs was administered to the mice (in incomplete Freund'sadjuvant) one month after priming. Repeat serology was performed a totalof two months after the initial priming dose. Hyperimmune sera from theboosted animals showed neutralizing ratios similar to the initialtesting.

In addition, mice vaccinated with VLPs based on BKV-Ib2 had serumantibody responses that robustly neutralized the cognate BKV-Ib2reporter pseudovirus but failed to effectively neutralize the BKC-Iapseudovirus (Table 2). This result confirms that genotypes BKV-Ia andBKV-Ib2 are distinct serotypes.

TABLE 2 Single immunization of mice with BKV variants Neutralizing titer(log) Ia Ib2 Ic II III IVb1 IVc2 IMMUNIZATION Ia 6.6 3.9 3.6 2.7 neg 2.62.0 Ib2 neg 4.5 3.6 neg neg 2.3 neg Ic 3.7 3.6 4.2 neg neg 2.8 neg II2.1 3.5 3.2 4.5 3.5 3.6 3.1 III neg 3.5 3.3 3.4 4.0 2.9 2.9 IVb1 neg 2.93.1 2.5 neg 4.6 4.1 IVc2 neg 3.4 3.5 3.0 2.2 3.8 4.2 All 7 types 4.0 3.94.1 4.6 4.0 4.7 4.7

Taken together, the data suggest that the neutralization assay has alarger quantitative dynamic range than ELISA and the neutralizationassay is on average about 10 times better at distinguishing BKVtype-specific titers (FIG. 1 , bottom panel). It is possible thatnon-neutralizing cross-reactive anti-BKV antibodies are being detectedin the ELISA assay but not in the neutralization assay. However, in thecontext of kidney transplantation, detection of type-specificneutralizing antibodies is more relevant, as only neutralizingantibodies would inhibit de novo infections with a new serotype orsuppress latent infections with an existing BKV serotype.

Example 3 BKV Titers in Healthy Adults

Sera from 48 healthy adults with a median age of 52.5 years wereassessed for reactivity to BKV-I and BKV-IV in ELISAs. Seroprevalence ofdifferent BKV types in these individuals is shown in Table 3. Of these,83% were seropositive for BKV-I (FIG. 3 , top panel), a figure similarto what has been reported in the literature (Egli et al., J. Infect.Dis. 199:837-846, 2009; Knowles et al., J. Med. Virol. 71:115-123,2003). The geometric mean titer for anti-BKV-I sera was 550 and itranged from a low of 60 to a high of 7100. In contrast, 65% ofvolunteers were seropositive for BKV-IV, but their geometric mean titerwas only 150, even when they had a similar range (60 to 17,000). In theBKV-IV ELISA, only 18% (9 sera) had a titer higher or equal to 500,while in the BKV-I ELISA 54% (26 sera) reached this titer. There wasalso a statistically significant correlation between BKV-I and BKV-IVtiters (Spearman r=0.69, p<0.0001). This correlation, along with thelower geometric mean titers and the knowledge that previous studies haveonly found 6-7% prevalence of BKV-IV DNA (Krumbholz et al., J. Med.Virol. 78:1588-1598, 2006), indicates that much of the BKV-IVseropositivity is attributable to cross-reactivity in the ELISA assay.The sera were therefore evaluated in the neutralization assay.

TABLE 3 Seroprevalence of BKV types in 48 healthy individuals Ia Ib2 IcII III IVb1 IVc2 % Prevalence 79 52 63 58 28 17 28

For the neutralization assays, serum samples were serially dilutedstarting at 1:100. This is the lowest naïve (rabbit) serum dilution thatis consistently devoid of non-specific neutralizing activity (Pastranaet al., PLoS Pathog. 5:e1000578, 2009). Therefore EC₅₀ values below thisdilution could not be accurately calculated and were arbitrarilydesignated to have an EC₅₀ of 100. Only 3 volunteers (6%) were negativefor BKV-I neutralization. In contrast, 37 (77%) were negative for BKV-IV(FIG. 3 , bottom panel). The geometric mean EC₅₀ titers for BKV-I werealso significantly higher (5100) than BKV-IV titers (180). There werethree individuals with titers of more than 60,000 for BKV-I that werecompletely negative for BKV-IV neutralization. In contrast to the ELISAresults, there was not a statistically significant correlation betweenthe subjects' BKV-I and BKV-IV neutralizing titers. There were twoindividuals with BKV-I neutralizing titers of >100,000 whose sera didnot detectably neutralize BKV-IV at the lowest tested dilution (1:100).This indicates that these individuals displayed BKV type specificityratios of at least 1,000. A comparison of ELISA values to neutralizationassay values is shown in FIG. 4 . Overall, the results for the humansera confirm the observations using murine sera, suggesting that theneutralization assays offer a significantly greater degree ofsensitivity and specificity for serological analysis of exposure toBKV-I and BKV-IV.

Example 4 BKV Type-Specific Seroconversion in Kidney TransplantRecipients

The anti-BKV-I and BKV-IV titers of 108 kidney transplant recipientswere determined. An archived set of sera collected at time points ofroughly 1, 4, 12, 26, and 52 weeks post-transplantation were tested inthe neutralization assay. Each sample was tested at four dilutions: 100,500, 5,000, and 50,000. Because of this lack of full serial dilution, amore stringent neutralization cutoff of 95% for individual data pointswas utilized. Neutralization assay results for individual subjects areshown in FIGS. 5 and 6 . At entry, 5 patients (5%) were seronegative(<95% neutralizing at the 1:100 serum dilution) in the BKV-Ineutralization assay (Table 4). In contrast, there were 53 initiallyBKV-IV seronegative subjects (49%). The patients were then assessed forseroconversion, defined as a change from seronegative at the first timepoint to at least 95% neutralization at the 1:500 serum dilution at anysubsequent time point. All of the 5 initially seronegative BKV-Ipatients seroconverted for BKV-I, and 23 (43%) of the initially BKV-IVseronegative patients seroconverted for BKV-IV (Table 4).

TABLE 4 Seroconversion of kidney transplant recipients Negative at entrySeroconversion Stringent Seroconversion (% total) (% initial negative)(% initial negative) BKV-I BKV-IV BKV-I BKV-IV BKV-I BKV-IV 5 (5%) 53(49%) 5 (100%) 23 (43%) 5 (100%) 12 (23%)

The average BKV type-specificity ratio for sera from immunized mice was1359 (FIG. 1 ). Two human subjects likewise showed type-specificityratios>1000 (FIG. 3 ). To address the possibility that BKV-IVneutralization might be partly attributable to cross-reactivity of hightiter antibody responses elicited by BKV-I, a more stringent definitionof seroconversion was applied, in which the ratio of the BKV-I titerversus the BKV-IV titer (or vice-versa) must be less than 1000 at leastone time point to be considered a clear type-specific seroconversionevent. Even with these stricter criteria, 12 (23%) of the BKV-IVnegative patients seroconverted within a year of transplantation (Table2). Based on the results shown in FIGS. 1 and 3, the occurrence of BKVtype-specificity ratios of 10 or less seems highly unlikely. Fivepatients (5%) underwent BKV-IV-specific seroconversion by the extremelystrict criterion of having a BKV-I to BKV-IV titer ratio<10.

On average, the patients' BKV-I and BKV-IV neutralizing titers bothincreased substantially by one year after renal transplantation (FIG. 7). In some instances, titer increases occurred even in patients whoshowed moderate neutralizing antibody titers at study entry (FIGS. 5 and6 ).

Example 5 BKV VP1 Protein Sequence Analysis

Full-length non-identical BKV VP1 peptide sequences available viaGenBank were aligned (FIG. 8 ). The GenBank Accession numbers utilizedto generate the alignment are provided in Table 5. Because it is notpossible to distinguish between Ia and Ib1 subtypes based on VP1 aminoacid sequences, BKV-Ia indicates genotypes Ia/Ib1 and BKV-Ib indicatesgenotype Ib2. It is also not possible to distinguish between BKV-IVsubtypes based on VP1 amino acid sequences. Therefore, BKV-IV indicatesgenotypes IV-b1/IV-c2.

TABLE 5 GenBank Accession numbers of aligned BKV VP1 sequences BKV typeGenBank Accession Number SEQ ID NO: Ia BAE96059 52 Ia CAA40239 53 IaBAF02957 54 Ia AAT47395 55 Ia AAT47401 56 Ia BAF42979 57 Ia ABI94689 58Ia ABI94671 59 Ia AAT47365 60 Ia AAT47389 61 Ia YP_717939 62 Ia AAT4737163 Ia CAA24307 64 Ia AAT47413 65 Ia AAT47419 66 Ia BAF42937 67 IaABI94725 68 Ia AFA41877 69 Ia AFA41907 70 Ia AAT47425 71 Ia AAT47431 72Ia CAA40243 73 Ia AEK21505 74 Ib ABI94623 75 Ib AFA41920 76 Ib ABI9461177 Ib BAF42907 78 Ib ABD04662 79 Ib ABI94713 80 Ib CBX88302 81 IbAFA41880 82 Ib BAF93325 83 Ib CAA40247 84 Ib CBX88314 85 Ib AB194617 86Ib BAF93319 87 Ib AAT47347 88 Ib BAF93283 89 Ib BAF03085 90 Ib AFA4190991 Ib BAF03097 92 Ib ABI94695 93 Ic BAE53660 94 Ic BAE53648 95 IcCAA40235 96 Ic BAI43588 97 Ic BAE53642 98 Ic BAG75361 99 Ic BAG75283 100Ic BAF76196 101 Ic ABI94635 102 Ic BAF02975 103 II BAF42901 104 IIBAF42925 105 II CAA79596 106 III BAF03017 107 III P14996 108 IIIAEO89615 109 IV BAF03115 110 IV BAE53654 111 IV BAF75138 112 IV BAE96077113 IV BAG75277 114 IV BAF75102 115 IV BAF03029 116 IV BAF75180 117 IVAFA41889 118 IV BAF75096 119 IV BAF75114 120 IV AFA41881 121 IV AFA41883122 IV AFA41885 123 IV BAG84476 124 IV BAF03035 125

With respect to the BKV-I consensus, BKV-IV isolates tend to carry avariety of substitutions: E61N, N62D, F66Y, K69R, S71T, N74T, D75A,S77D, E82D, Q117K, H139N, 1178V, F225Y, A284P, R340Q, K353R, and L362V.Mapping of these BKV-I/BKV-IV variant residues onto homologous positionsin the X-ray crystal structures of JCV (Neu et al., Cell Host Microbe8:309-319, 2(10) and SV40 (Stehle et al., Structure 4:165-182, 1996)suggested that, with the exception of positions 117, 225, 2.84, and 340,each of these BKV-I/BKV-IV variant residues is likely to be exposed onthe exterior surface of the capsid. With the exception of residues 353and 362, which are exposed along the floor of the canyons betweencapsomer knobs, all the exposed variations map to sites on the apicalsurface and apical rim of the capsomer knob. Many of the variations areadjacent to residues predicted to be involved in binding the cellularglycolipids that serve as receptors during BKV infectious entry (Duganet al., J. Virol. 81:11798-11808, 2007; Low et al., J. Virol.80:13614366, 2006). This is consistent with the idea that BKV-I/BKV-IVvariations may alter epitopes recognized by antibodies that neutralizeinfectivity via steric occlusion of the receptor binding site.

In addition to the differences between BKV-1 and BKV-IV, severalpositions differ stereotypically among BKV-I subtypes. For example, BKVsubtype Ib-2 isolates tend to carry V42L, P82D, D175E, V210I, R340K, andL362V differences, with respect to subtypes Ia and Ib-1. Likewise,subtype Ic isolates frequently carry E20D, F225L, and R340K differences.Although these intra-genotype-I surface variations are chemicallysubtle, without being hound by theory, it is possible that thedifferences reflect selective pressure to escape neutralizingantibodies,

In human subjects, it was found that 6 of 48 subjects with BKV-Ianeutralizing antibodies were not able to neutralize a BKV-Ib2 isolate(including VP1 polypeptide with the amino acid sequence of SEQ ID NO:14), Analysis of mutant pseudoviruses that are recombinant chimeras oftypes Ia and Ib2, identified key amino acid residues that allow the Ib2isolate to escape from Ia-neutralizing antibody response. The variationswere in the VP1 BC loop and included E73K and E82D (Ia to Ib2variations), Several other Ib2 variants and genotype Ic BC loopvariations were partially resistant to Ia-neutralizing human sera. Theseadditional variants included E73Q, S77N, E82Q, or combinations of thesevariations. These results suggest that an optimal BKV vaccine shouldinclude at least BKV-Ia and BKV-Ib2 VP1 polypeptides, in order to elicitantibodies capable of neutralizing all BKV-I variants. Furthermore,validation of a candidate vaccine should include screening serum fromvaccinated subjects for neutralization of multiple BKV-I subtypes (suchas at least BKV-Ia and BKV-Ib2 subtypes).

Example 6 Additional BKV VP1 Polypeptides

Additional BKV serotypes may remain to be discovered and fullysequenced. GenBank accession numbers CCF70703-CCF70735 report a portionof the VP1 protein encompassing the BC and EF loops. Some of thesesequences contain previously unknown variations in the BC and EF loops.In some instances, the sequence fragments are more divergent from allpublished BKV VP1 sequences than BKV-I is from BKV-IV (FIG. 9 ). Basedon the an alignment of these additional sequences with BKV-Ia, BKV-Ib,BKV-Ic, BKV-II, BKV-III, BKV-IVb1, and BKV-IVc2 VP1 sequences (FIG. 10), accession numbers CCF70725 (SEQ ID NO: 154), CCF70727 (SEQ ID NO:153), CCF70729 (SEQ ID NO: 158), and CCF70730 (SEQ ID NO: 157) appear torepresent portions of distinct BKV serotypes. The GenBank Accessionnumbers utilized to generate the alignment are provided in Table 6. Theserological distinctiveness of these recently reported sequences can bedetermined, for example by isolating the remainder of these VP1sequences and using the methods described in Examples 1 and 2.Polypeptides comprising these additional BKV VP1 polypeptides can beutilized in the disclosed methods and compositions. In some cases, thedisclosed methods and compositions include one or more of SEQ ID NOs:126-158.

TABLE 6 GenBank Accession Nos. of additional partial BKV VP1 sequencesGenBank Accession No. SEQ ID NO: CCF70703 126 CCF70704 127 CCF70705 128CCF70706 129 CCF70707 130 CCF70708 131 CCF70709 132 CCF70710 133CCF70711 134 CCF70712 135 CCF70713 136 CCF70714 137 CCF70715 138CCF70716 139 CCF70717 140 CCF70718 141 CCF70719 142 CCF70720 143CCF70722 144 CCF70723 145 CCF70724 146 CCF70726 147 CCF70728 148CCF70732 149 CCF70733 150 CCF70734 151 CCF70735 152 CCF70727 153CCF70725 154 CCF70731 155 CCF70721 156 CCF70730 157 CCF70729 158

Example 7 Methods of Eliciting an Immune Response to BKV

This example provides exemplary methods for eliciting an immune responseto one or more BKV serotypes in a subject. However, one of ordinaryskill in the art will appreciate that methods that deviate from thesespecific methods can also be used to successfully elicit an immuneresponse to BKV in a subject.

In particular examples, the method includes selecting a subject in needof enhanced immunity to BKV. Subjects in need of enhanced immunity toBKV include individuals who are immunocompromised and individuals whohave had or are candidates for organ transplantation, for example arenal transplant or bone marrow transplant. Subjects in need of enhancedimmunity to BKV also include individuals who are seronegative for atleast one BKV serotype.

Subjects selected for treatment are administered a therapeuticallyeffective amount of a disclosed immunogenic composition. In someexamples, a therapeutically effective amount of one or more BKV-I capsidpolypeptides (or fragments thereof) or one or more polynucleotidesencoding the BKV-I capsid polypeptides and a therapeutically effectiveamount of one or more BKV-IV capsid polypeptides (or fragments thereof)or one or more polynucleotides encoding the BKV-IV capsid polypeptidesis administered to the subject at doses of about 0.1 μg to 10 mg of eachBKV capsid polypeptide or polynucleotide encoding the polypeptide orabout 20-40 μg VLP per type for pentamers. However, the particular dosecan be determined by a skilled clinician. The disclosed BKV capsidpolypeptides (or a fragment thereof) or polynucleotide encoding the BKVcapsid polypeptides or fragment thereof can be administered in one orseveral doses, for example continuously, daily, weekly, or monthly. Whenadministered sequentially, the time separating the administration can beseconds, minutes, hours, days, or even weeks.

The mode of administration can be any used in the art, including but notlimited to subcutaneous or intramuscular administration. The amount ofagent administered to the subject can be determined by a clinician, andmay depend on the particular subject treated. Specific exemplary amountsare provided herein (but the disclosure is not limited to such doses).

The development of immune response (such as development of antibodies,such as neutralizing antibodies) in a subject is monitored at timepoints following administration of the immunogenic composition. Methodsof detecting antibodies in a sample (such as a blood or serum sample)include those known in the art, for example, ELISA methods. In someexamples, the development of neutralizing antibodies to a BKV-Ia and/orBKV-Ib1 subtype and development of neutralizing antibodies to BKV-Ib2subtype are monitored.

Example 8 Methods of Treating or Inhibiting PVAN

This example provides exemplary methods for treating or inhibiting PVANin a subject. However, one of ordinary skill in the art will appreciatethat methods that deviate from these specific methods can also be usedto successfully treat or inhibit PVAN in a subject.

In particular examples, the method includes selecting a subject having,thought to have, or at risk of having PVAN. Subjects having or thoughtto have PVAN include those with >10 inclusion bearing epithelial cells(“decoy cells”) in a urine sample per 10 high power fields, >10⁷ BKVcopies per 10 mL urine, or histopathological identification of viralalterations in a renal biopsy. Subjects at risk of PVAN include thosewho have had or are candidates for organ transplantation (such as renaltransplant or bone marrow transplant), and immunocompromisedindividuals. In some examples, subjects who are candidates for renaltransplant are selected. The selected subject can be a subject who doesnot have BKV-IV neutralizing antibodies.

Subjects selected for treatment are administered a therapeuticallyeffective amount of a disclosed immunogenic composition. In someexamples, a therapeutically effective amount of one or more BKV-IVcapsid polypeptides or a polynucleotide encoding the BKV-IV capsidpolypeptide(s) is administered to the subject at doses of about 0.1 μgto 10 mg of each BKV-IV capsid polypeptide or polynucleotide encodingthe polypeptide or about 20-40 μg VLP per type for pentamers. However,the particular dose can be determined by a skilled clinician. Thedisclosed BKV-IV capsid polypeptides (or a fragment thereof) orpolynucleotide encoding the BKV-IV capsid polypeptides or fragmentthereof can be administered in one or several doses, for examplecontinuously, daily, weekly, or monthly, with at least one dose at least2 weeks prior to transplant. When administered sequentially, the timeseparating the administration can be seconds, minutes, hours, days, oreven weeks.

The mode of administration can be any used in the art, including but notlimited to subcutaneous or intramuscular administration. The amount ofagent administered to the subject can be determined by a clinician, andmay depend on the particular subject treated. Specific exemplary amountsare provided herein (but the disclosure is not limited to such doses).

The development of immune response (such as development of antibodies,such as neutralizing antibodies) in a subject is monitored at timepoints following administration of the immunogenic composition. Methodsof detecting antibodies in a sample (such as a blood or serum sample)include those known in the art, for example, ELISA methods. A renaltransplant is performed after an immune response is detected. Thesubject is also monitored for development of PVAN, for example bytesting for presence of virus in urine or renal biopsy.

Example 9 Methods of Identifying a Renal Transplant Donor

This example provides exemplary methods for identifying a renaltransplant donor, such as an individual who does not have serumantibodies capable of neutralizing one or more BKV types. However, oneof ordinary skill in the art will appreciate that methods that deviatefrom these specific methods can also be used to successfully identify arenal transplant donor.

A blood sample is collected from a subject who is considered a possiblerenal transplant donor. Serum is prepared from the sample and serialdilutions of the serum are prepared (e.g., ten serial dilutions offour-fold ranging from 1:100 to 2.6×10⁷). The diluted serum is mixedwith a diluted BKV reporter vector carrying a reporter plasmid encodingGaussia princeps luciferase under the control of a human elongationfactor 1 alpha promoter and incubated on ice or at 4° C. for 1 hour.Then, the virus/serum mixture is added to 293TT cells plated in a96-well plate at 3×10⁴ cells/well. After 3 days, 25 μl of conditionedmedium is collected and transferred to a luminometry plate. Gaussialuciferase substrate (50 μl NEB Gaussia luciferase assay kit substrate)is added and light emission is detected using a luminometer. Effectiveconcentration 50% (EC₅₀) is calculated for the serum dilution series. AnEC₅₀ of less than 100 indicates that the subject does not have BKV-IVneutralizing antibodies. A subject who does not have BKV-IV neutralizingantibodies is selected as a renal transplant donor.

A candidate renal transplant recipient can also be screened for thepresence of BKV-IV neutralizing antibodies as described above. Acandidate renal transplant recipient who is negative for BKV-IVneutralizing antibodies is considered a good candidate for receiving akidney from a renal transplant donor who is negative for BKV-IVneutralizing antibodies.

Similar screening can be done for the presence of other BKVtype-specific neutralizing antibodies in potential renal transplantdonors and/or recipients. A potential renal transplant donor who doesnot have particular type-specific (such as BKV-I, BKV-II, or BKV-III)neutralizing antibodies is identified and selected as a renal transplantdonor, particularly for a renal transplant recipient who also does nothave the same type-specific neutralizing antibodies.

In view of the many possible embodiments to which the principles of thedisclosure may be applied, it should be recognized that the illustratedembodiments are only examples and should not be taken as limiting thescope of the invention. Rather, the scope of the invention is defined bythe following claims. We therefore claim as our invention all that comeswithin the scope and spirit of these claims.

We claim:
 1. A method of treating or inhibiting BKpolyomavirus-associated nephropathy or BK polyomavirus-associatedhemorrhagic cystitis in a subject, comprising administering to a subjectin need of treatment for or inhibition of BK polyomavirus-associatednephropathy or BK polyomavirus-associated hemorrhagic cystitis atherapeutically effective amount of at least one isolated BKV serotypeIV (BKV-IV) capsid polypeptide or a nucleic acid encoding the at leastone BKV-IV capsid polypeptide, thereby treating or inhibiting the BKpolyomavirus-associated nephropathy or the BK polyomavirus-associatedhemorrhagic cystitis.
 2. The method of claim 1, further comprisingadministering to the subject a therapeutically effective amount of: atleast one isolated BKV serotype I (BKV-I) capsid polypeptide or anucleic acid encoding the at least one BKV-I capsid polypeptide; atleast one isolated BKV serotype II (BKV-II) capsid polypeptide or anucleic acid encoding the at least one BKV-II capsid polypeptide; atleast one isolated BKV serotype III (BKV-III) capsid polypeptide or anucleic acid encoding the at least one BKV-III capsid polypeptide; atleast one isolated JC polyomavirus capsid polypeptide or a nucleic acidencoding the at least one JC polyomavirus capsid polypeptide; or acombination of two or more thereof.
 3. The method of claim 1, furthercomprising administering an adjuvant to the subject.
 4. The method ofclaim 1, wherein the subject is an immunocompromised subject or asubject who has been treated with or is a candidate for treatment withan immunosuppressant.
 5. The method of claim 1, wherein the subject is asubject who has received or is a candidate for an organ transplant. 6.The method of claim 5, wherein the subject has received or is acandidate for a kidney transplant or a bone marrow transplant.
 7. Themethod of claim 5, wherein the subject is a candidate for an organtransplant and the therapeutically effective amount of the at least oneBKV-IV capsid polypeptide or nucleic acid encoding the at least oneBKV-IV capsid polypeptide is administered to the subject a sufficienttime prior to the organ transplant to produce an immune response in thesubject.
 8. The method of claim 1, wherein the subject does not haveBKV-IV neutralizing antibodies.
 9. The method of claim 1, wherein thesubject is a subject in need of treatment for or inhibition of BKpolyomavirus-associated nephropathy.
 10. The method of claim 9, furthercomprising selecting the subject in need of treatment for or inhibitionof BK polyomavirus-associated nephropathy.